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系統識別號 U0007-0607201010523200
論文名稱(中文) 含氮之[6,5] 雜環類緣物之合成與抗癌活性研究
論文名稱(英文) Synthesis of Nitrogen-containing [6,5]-fused heterocycles as Anticancer Agents
校院名稱 臺北醫學大學
系所名稱(中) 藥學研究所
系所名稱(英) Graduate Institute of Pharmacy
學年度 99
學期 2
出版年 99
研究生(中文) 沈柏榕
學號 M301097019
學位類別 碩士
語文別 中文
口試日期 2010-06-03
論文頁數 237頁
口試委員 指導教授-劉景平
委員-林仁混
委員-林美香
關鍵字(中) 含氮之[6,5] 雜環
組織蛋白去乙醯酶
抑制劑
關鍵字(英) Nitrogen-containing [6,5]-fused heterocycles
Histone deacetylase inhibitors
學科別分類
中文摘要 組織蛋白去乙醯酶抑制劑(Histone deacetylase inhibitor, HDACi)目前廣泛的用於治療癌症,其中已有多種藥物上市,例如默克藥廠的小分子化合物ZolinzaR(vorinostat, SAHA, FDA approved for treatment of refractory cutaneous T-call lymphoma in 2006)和Gloucester藥廠的RomidepsinR(FK-228, FDA approved for treatment of refractory cutaneous T-call lymphoma in 2009),以上都證明此類藥物為治療癌症之有效策略。
本實驗室觀察相關HDACi後發現,N-hydroxyacrylamide和benzamide等官能基在抑制活性上扮演重要角色;含氮之[6,5] 雜環,例如indole,在許多抗癌小分子化合物中為重要骨架,因此本實驗室決定探討含氮之[6,5] 雜環在其N位以苯磺胺類或是苯烷基連結,導入N-hydroxyacrylamide和benzamide官能基,合成二系列化合物,進一步了解HDAC抑制活性與結構之間關係。
實驗室合成出的二系列化合物對口腔上皮細胞癌KB cell line做生物活性試驗,目前已知的資料中12d、17d、24c、28b有相似的活性,其中以24c IC50 = 604.6 nM最好。目前其他化合物活性實驗仍在進行中,此後將會作HDAC抑制活性試驗與結構修飾。
英文摘要 Histone deacetylase inhibitors (HDACi) have been used for anticancer drugs broadly. Some of them were approved by FDA, for example ZolinzaR and RomidepsinR for treatment of refractory cutaneous T-call lymphoma. Therefore, to target histone deacetylase provides a potential methodology to develop potent anticancer agents.
To cording to the structures of HDACi, our laboratory found benzamide and N-hydroxyacrylamide important to HDAC inhibition activity. Besides, we observed some small molecular anticancer drugs containing nitrogen-containing [6,5]-fused heterocycles as major composition, for instance indole . So we introduced benzenesulfonamide or benzene motif on the N position of the nitrogen-containing [6,5]-fused heterocycles as the linker region and utilized N-hydroxyacrylamide and benzamide group for chelating region to discuss the relationship between structures and HDAC inhibition activity.
The two series of compounds are going to test KB cell line. According to the result, compound 12d, 17d, 24c and 28b have similar activity. Compound 24c possess the most potent inhibitory activity (IC50 = 604.6 nM). The evaluation of biological activities is still in progress. The further structural optimization and HDAC inhibition activity tests are going to be investigated.
論文目次 附圖目錄 9
中文摘要 15
英文摘要 16
研究背景 17
實驗目的與設計 28
生物活性結果與討論 40
結論 41
實驗儀器 44
試藥與試劑 46
試藥與試劑簡寫表 49
合成步驟 50
indoline (1) 50
7-chloro-4-azaindole (2) 51
4-azaindole (3) 52
7-chloro-6-azaindole (4) 53
6-azaindole (5) 54
5-azaindole (6) 55
3-(chlorosulfonyl)benzoate (7) 56
methyl 3-(1H-indol-1-ylsulfonyl)benzoate (8a) 57
3-(1H-indol-1-ylsulfonyl)benzaldehyde (8b) 58
(E)-tert-butyl 3-(3-(1H-indol-1-ylsulfonyl)phenyl)acrylate (8c) 59
(E)-3-(3-(1H-indol-1-ylsulfonyl)phenyl)acrylic acid (8d) 60
(E)-3-(3-(1H-indol-1-ylsulfonyl)phenyl)-N-hydroxyacrylamide (8e) 61
methyl 3-(indolin-1-ylsulfonyl)benzoate (9a) 62
3-(indolin-1-ylsulfonyl)benzaldehyde (9b) 63
(E)-tert-butyl 3-(3-(indolin-1-ylsulfonyl)phenyl)acrylate (9c) 64
(E)-3-(3-(indolin-1-ylsulfonyl)phenyl)acrylic acid (9d) 65
(E)-N-hydroxy-3-(3-(indolin-1-ylsulfonyl)phenyl)acrylamide (9e) 66
1-(3-bromophenylsulfonyl)-1H-pyrrolo[3,2-b]pyridine (10a) 67
(E)-tert-butyl-3-(3-(1H-pyrrolo[3,2-b]pyridin-1-ylsulfonyl)phenyl)acrylate (10b) 68
(E)-3-(3-(1H-pyrrolo[3,2-b]pyridin-1-ylsulfonyl)phenyl)acrylic acid (10c) 69
(E)-3-(3-(1H-pyrrolo[3,2-b]pyridin-1-ylsulfonyl)phenyl)-N-hydroxyacrylamide (10d) 70
1-(3-bromophenylsulfonyl)-1H-pyrrolo[2,3-c]pyridine (11a) 71
(E)-tert-butyl-3-(3-(1H-pyrrolo[2,3-c]pyridin-1-ylsulfonyl)phenyl)acrylate (11b) 72
(E)-3-(3-(1H-pyrrolo[2,3-c]pyridin-1-ylsulfonyl)phenyl)acrylic acid (11c) 73
(E)-3-(3-(1H-pyrrolo[2,3-c]pyridin-1-ylsulfonyl)phenyl)-N-hydroxyacrylamide (11d) 74
1-(3-bromophenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (12a) 75
(E)-tert-butyl-3-(3-(1H-pyrrolo[2,3-b]pyridin-1-ylsulfonyl)phenyl)acrylate (12b) 76
(E)-3-(3-(1H-pyrrolo[2,3-b]pyridin-1-ylsulfonyl)phenyl)acrylic acid (12c) 77
(E)-3-(3-(1H-pyrrolo[2,3-b]pyridin-1-ylsulfonyl)phenyl)-N-hydroxyacrylamide (12d) 78
1-(3-bromophenylsulfonyl)-2,3-dihydro-1H-pyrrolo[2,3-b]pyridine (13a) 79
(E)-tert-butyl-3-(3-(2,3-dihydro-1H-pyrrolo[2,3-b]pyridin-1-ylsulfonyl)phenyl)acrylate (13b) 80
(E)-3-(3-(2,3-dihydro-1H-pyrrolo[2,3-b]pyridin-1-ylsulfonyl)phenyl)acrylic acid (13c) 81
(E)-3-(3-(2,3-dihydro-1H-pyrrolo[2,3-b]pyridin-1-ylsulfonyl)phenyl)-N-hydroxyacrylamide (13d) 82
1-(3-bromophenylsulfonyl)-1H-indazole (14a) 83
(E)-tert-butyl 3-(3-(1H-indazol-1-ylsulfonyl)phenyl)acrylate (14b) 84
(E)-3-(3-(1H-indazol-1-ylsulfonyl)phenyl)acrylic acid (14c) 85
(E)-3-(3-(1H-indazol-1-ylsulfonyl)phenyl)-N-hydroxyacrylamide (14d) 86
1-(4-bromophenylsulfonyl)-1H-pyrrolo[3,2-b]pyridine (15a) 87
(E)-tert-butyl-3-(4-(1H-pyrrolo[3,2-b]pyridin-1-ylsulfonyl)phenyl)acrylate (15b) 88
(E)-3-(4-(1H-pyrrolo[3,2-b]pyridin-1-ylsulfonyl)phenyl)acrylic acid (15c) 89
(E)-3-(4-(1H-pyrrolo[3,2-b]pyridin-1-ylsulfonyl)phenyl)-N-hydroxyacrylamide (15d) 90
1-(4-bromophenylsulfonyl)-1H-pyrrolo[2,3-c]pyridine (16a) 91
(E)-tert-butyl-3-(4-(1H-pyrrolo[2,3-c]pyridin-1-ylsulfonyl)phenyl)acrylate (16b) 92
(E)-3-(4-(1H-pyrrolo[2,3-c]pyridin-1-ylsulfonyl)phenyl)acrylic acid (16c) 93
(E)-3-(4-(1H-pyrrolo[2,3-c]pyridin-1-ylsulfonyl)phenyl)-N-hydroxyacrylamide (16d) 94
1-(4-bromophenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (17a) 95
(E)-tert-butyl-3-(4-(1H-pyrrolo[2,3-b]pyridin-1-ylsulfonyl)phenyl)acrylate (17b) 96
(E)-3-(4-(1H-pyrrolo[2,3-b]pyridin-1-ylsulfonyl)phenyl)acrylic acid (17c) 97
(E)-3-(4-(1H-pyrrolo[2,3-b]pyridin-1-ylsulfonyl)phenyl)-N-hydroxyacrylamide (17d) 98
1-(4-bromophenylsulfonyl)-2,3-dihydro-1H-pyrrolo[2,3-b]pyridine (18a) 99
(E)-tert-butyl-3-(4-(2,3-dihydro-1H-pyrrolo[2,3-b]pyridin-1-ylsulfonyl)phenyl)acrylate (18b) 100
(E)-3-(4-(2,3-dihydro-1H-pyrrolo[2,3-b]pyridin-1-ylsulfonyl)phenyl)acrylic acid (18c) 101
(E)-3-(4-(2,3-dihydro-1H-pyrrolo[2,3-b]pyridin-1-ylsulfonyl)phenyl)-N-hydroxyacrylamide (18d) 102
1-(4-bromophenylsulfonyl)-1H-indazole (19a) 103
(E)-tert-butyl 3-(4-(1H-pyrazolo[3,4-b]pyridin-1-ylsulfonyl)phenyl) -acrylate (19b) 104
(E)-3-(4-(1H-indazol-1-ylsulfonyl)phenyl)acrylic acid (19c) 105
(E)-3-(4-(1H-indazol-1-ylsulfonyl)phenyl)-N-hydroxyacrylamide (19d) 106
4-(indolin-1-ylsulfonyl)benzoic acid (20a) 107
N-(2-aminophenyl)-4-(indolin-1-ylsulfonyl)benzamide (20b) 108
methyl 4-((1H-pyrrolo[3,2-b]pyridin-1-yl)methyl)benzoate (21a) 109
4-((1H-pyrrolo[3,2-b]pyridin-1-yl)methyl)benzoic acid (21b) 110
4-((1H-pyrrolo[3,2-b]pyridin-1-yl)methyl)-N-(2-aminophenyl)benzamide (21c) 111
methyl 4-((1H-pyrrolo[3,2-c]pyridin-1-yl)methyl)benzoate (22a) 112
4-((1H-pyrrolo[3,2-c]pyridin-1-yl)methyl)benzoic acid (22b) 113
4-((1H-pyrrolo[3,2-c]pyridin-1-yl)methyl)-N-(2-aminophenyl)benzamide (22c) 114
methyl 4-((1H-pyrrolo[2,3-c]pyridin-1-yl)methyl)benzoate (23a) 115
4-((1H-pyrrolo[2,3-c]pyridin-1-yl)methyl)benzoic acid (23b) 116
4-((1H-pyrrolo[2,3-c]pyridin-1-yl)methyl)-N-(2-aminophenyl)benzamide (23c) 117
methyl 4-((1H-pyrrolo[2,3-b]pyridin-1-yl)methyl)benzoate (24a) 118
4-((1H-pyrrolo[2,3-b]pyridin-1-yl)methyl)benzoic acid (24b) 119
4-((1H-pyrrolo[2,3-b]pyridin-1-yl)methyl)-N-(2-aminophenyl)benzamide (24c) 120
methyl 4-((1H-indazol-1-yl)methyl)benzoate (25a) 121
4-((1H-indazol-1-yl)methyl)benzoic acid (25b) 122
4-((1H-indazol-1-yl)methyl)-N-(2-aminophenyl)benzamide (25c) 123
methyl 4-((1H-indol-1-yl)methyl)benzoate (26a) 124
4-((1H-indol-1-yl)methyl)benzoic acid (26b) 125
4-((1H-indol-1-yl)methyl)-N-(2-aminophenyl)benzamide (26c) 126
4-(indolin-1-ylmethyl)benzoic acid (27a) 127
N-(2-aminophenyl)-4-(indolin-1-ylmethyl)benzamide (27b) 128
4-((2,3-dihydro-1H-pyrrolo[2,3-b]pyridin-1-yl)methyl)benzoic acid (28a) 129
N-(2-aminophenyl)-4-((2,3-dihydro-1H-pyrrolo[2,3-b]pyridin-1-yl)methyl)benzamide (28b) 130
附圖部分 131
參考文獻 230
參考文獻 1. 行政院衛生署http://www.doh.gov.tw/
2. Davie, J. R. Covalent modifications of histones: expression from chromatin templates. Curr. Opin. Genet. Dev. 1998, 8, 173-178.
3. Strahl, B. D.; Allis, C. D. The language of covalent histone modifications. Nature. 2000, 403, 41-45.
4. Roth, S. Y.; Denu, J. M.; Allis, C. D. Histone acetyltransferases. Annu. Rev. Biochem. 2001, 70, 81-120.
5. Andrew, A. L.; Bruce A. C. Histone deacetylase inhibitors in cancer therapy. J. Clin. Oncol. 2009, 27, 5459-5468.
6. Bolden, J. E.; Peart, M. J.; Johnstone, R. W. Anticancer activities of histone deacetylase inhibitors. Nat Rev Drug Discov. 2006, 5, 769-784.
7. Ito, K.; Barnes, P. J.; Adcock, I. M. Glucocorticoid receptor recruitment of histone deacetylase 2 inhibits interleukin-1β induced histone H4 acetylation on lysines 8 and 12. Mol. Cell Biol. 2000, 20, 6891-6903.
8. Cai, R. L.; Yan, Y. N.; Maria, A. C.; Hong, X.; Dalia, C. HDAC1, a histone deacetylase, forms a complex with Hus1 and Rad9, two G2/M checkpoint Rad proteins. J. Biol. Chem. 2000, 275, 27909-27916.
9. Robertson, K. D. ; Slimane, A-S-A.; Tomoki, Y.; Paul, A. W.; Peter, L. J.; Alan, P. W. DNMT1 forms a complex with Rb, E2F1 and HDAC1 and represses transcription from E2Fresponsive promoters. Nature Genet. 2000, 25, 338-342.
10. Smirnov, D. A.; Hou, S.; Ricciardi, R. P. Association of histone deacetylase with COUP-TF in tumorigenic Ad12- transformed cells and its potential role in shut-off of MHCclass I transcription. Virology. 2000, 268, 319-328.
11. Cress, W. D.; Seto, E. Histone deacetylases, transcriptional control and cancer. J. Cell. Physiol. 2000, 184, 1-16.
12. Ng, H-H.; Bird, A. DNA methylation and chromatin modification. Curr. Opin. Genet. Dev. 1999, 9, 158-63.
13. Kurkjian1, C.; Kummar S.; Murgo, A. J. DNA methylation: its role in cancer development and therapy. Curr. Probl. Cancer. 2008, 32, 187-235.
14. Yang, X.; Yan, L.; Davidson, N. E., DNA methylation in breast cancer. Endocr. Relat. Cancer. 2001, 8, 115-127.
15. Miller, T. A.; Witter, D. J.; Belvedere, S. Histone deacetylase inhibitors. J. Med. Chem. 2003, 46, 5097-5116.
16. Gottlicher, M.; Minucci, S.; Kramer, H. O.; Schimpf, A.; Giavara, S.; Sleeman, P. J.; Coco, F.L.; Nervi, C.; Pelicci, P. G.; Heinzel, T. Valproic acid a novel class of HDAC inhibitorsinducing differentiation of transformed cells. EMBO. J. 2001, 20, 6969-6978.
17. Atmaca , A.; Al-Batran, S-E.; Maurer, A.; Neumann, A.; Heinzel, T.; Hentsch, B.; Schwarz, S. E.; Hovelmann, S.; Gottlicher, M.; Knuth, A.; Jager, E. Valproic acid (VPA) in patients with refractory advanced cancer: a dose escalating phase I clinical trial. Br. J. Cancer. 2007, 97, 177-182.
18. Sharma, S.; Symanowski, J.; Wong, B.; Dino, P.; Manno, P.; Vogelzang, N. A phase II clinical trial of oral valproic acid in patients with castration-resistant prostate cancers using an intensive biomarker sampling strategy. Transl. Oncol. 2008, 1, 141-147.
19. Itazaki, H.; Nagashima, K.; Sugita, K.; Yoshida, H.; Kawamura, Y.; Yasuda, Y.; Matsumoto, K.; Ishii, K.; Uotani, N.; Nakai, H.; Terui, A.; Yoshimatsu, S. Isolation and structural elucidation of new cyclotetrapeptides, Trapoxins A And B, having detransformation activities as antitumor agents. J. Antibiot. 1990, 43, 1524-1532.
20. Shute R.E.; Dunlap, B.; Rich, D. H. Analogues of the cytostatic and antimitogenic agents chlamydocin and HC-toxin: synthesis and biological activity of chloromethyl ketone and diazomethyl ketone functionalized cyclic tetrapeptides. J. Med. Chem. 1987, 30, 71-78.
21. Kijima, M.; YoshidaS, M.; Siugitae K.; Horinouchi, S.; Beppu, T. Trapoxin, an antitumor cyclic tetrapeptide, Is an irreversible inhibitor of mammalian histone deacetylase. J. Biol. Chem. 1993, 268, 22429-22435.
22. Suzuki, T.; Ando, T.; Tsuchiya, K.; Fukazawa, N. Synthesis and histone deacetylase inhibitory activity of new benzamide derivatives. J. Med. Chem. 1999, 42, 3001-3003.
23. Kato, Y.; Yoshimura, K.; Shin, T.; Verheul, H.; Hammers, H.; Sanni, T. B.; Salumbides, B. C.; Van Erp, K.; Schulick, R.; Pili, R. Synergistic in vivo antitumor effect of the histone deacetylase inhibitor MS-275 in combinationwith interleukin 2 in a murine model of renal cell carcinoma. Clin. Cancer Res. 2007, 13, 4538-4546
24. Khandelwal, A.; Gediya, L.K.; Njar, V. C. O. MS-275 synergistically enhances the growth inhibitory effects of RAMBA VN/66-1 in hormone-insensitive PC-3 prostate cancer cells and tumours. Br. J. Cancer. 2008, 98, 1234-1243.
25. Baradari, V.; Hopfner, M.; Huether, A.; Schuppan, D.; Scherubl, H. Histone deacetylase inhibitor MS-275 alone or combined with bortezomib or sorafenib exhibits strong antiproliferative action in human cholangiocarcinoma cells. World J. Gastroenterol. 2007, 13, 4458-4466.
26. Gore, Lia.; Rothenberg, L. M.; O’Bryant, L. C.; Schultz, M. K.; Sandler, B.; Coffin, D. A.; McCoy, Candice.; Schott, A.; Scholz, C.; Eckhardt, S. Gail. A phase I and pharmacokinetic study of the oral histone deacetylase inhibitor,MS-275, in patients with refractory solid tumors and lymphomas. Clin. Cancer Res. 2008, 14, 4517-4525.
27. Zhou, N.; Moradei, O.; Raeppel, S.; Leit, S.; Frechette, S.; Gaudette, F.; Paquin, I.; Bernstein, N.; Bouchain, G.; Vaisburg, A.; Jin, Z. Gillespie, J.; Wang, J.; Fournel, M.; Yan, T. P.; Trachy-Bourget, M-C.; Kalita, A.; Lu, A.; Rahil, J.; MacLeod, R. A.; Li, Z.; Besterman, M. J.; Delorme, D. Discovery of N-(2-aminophenyl)-4-[(4-pyridin-3- lpyrimidin-2-ylamino)methyl]benzamide (MGCD0103),an orally active histone deacetylase inhibitor. J. Med. Chem. 2008, 51, 4072-4075.
28. G, G-M.; Yang, A.; Klimek, V.; Luger, S.; Newsome, W.; Berman, N.; Patterson, T.; Maroun, C.; Li, Z.; Ward, R.; Martell, R. E. Phase I/II study of the novel oral isotype-selective histone deacetylase (HDAC) inhibitor MGCD0103 in combination with azacitidine in patients with high risk myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). J. Clin. Oncol. 2007, 25, 7062.(abstr.).
29. Blum, A. K.; Advani, A.; Fernandez, L.; Van Der Jagt, R.; Brandwein, J.; Kambhampati, S.; Kassis, J.; Davis, M.; Bonfils, C.; Dubay, M.; Dumouchel, J.; Drouin, M.; Lucas, M. D.; Marte, E. R.; Byrd, J. C. Phase II study of the histone deacetylase inhibitor MGCD0103 in patients with previously treated chronic lymphocytic leukemia. Br. J. Haematol. 2009, 147, 507-514.
30. Marks, P. A,; Rifkind, R. A.; Jursic, B. Novel potent inducers of terminal differentiation and methods of use thereof. PCT Int. Appl. WO 93107148. (abstr.).
31. U.S. Food and Drug Administration. http://www.fda.gov/
32. Yoshida, M.; Nomura, S.; Beppu, T. Effects of trichostatins on differentiation of murine erythroleukemia cells. Cancer Res. 1987, 47, 3688-3691.
33. Yoshida, M.; Kijima, M.; Akita, M.; Beppu, T. Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro by trichostatin A. J. Biol. Chem. 1990, 265, 17174-17179.
34. Atadja, P.; Lin, G.; Kwon, P.; Trogani, N.; Heather, W.; Hsu, M.; Yeleswarapu, L.; Chandramouli, N.; Perez, L.; Versace, R.; Wu, A.; Sambucetti, L.; Lassota, P.; Cohen, D.;Bair, K.; Wood, A.; Remiszewski, S. Selective growth inhibition of tumor cells by a novel histone deacetylase inhibitor NVP-LAQ824 Cancer Res. 2004, 64, 689-695.
35. Catley, L.; Weisberg, E.; Tai, Y-T.; Atadja, P.; Remiszewski, S.; Hideshima, T.; Mitsiades, N.; Shringarpure, R.; LeBlanc, R.; Chauhan, D.; Munshi, NC.; Schlossman, R.; Richardson, P.; Griffin, J.; Anderson, KC. NVP-LAQ824 is a potent novel histone deacetylase inhibitor with significant activity against multiple myeloma. Blood. 2003, 102, 2615-22.
36. Nimmanapalli, R.; Fuino, L,; Bali, P,; Gasparetto, M.; Glozak, M.; Tao, J.; Moscinski, L.; Smith, C.; Wu, J.; Jove, R.; Atadja, P.; Bhalla, K. Histone deacetylase inhibitor LAQ824 both lowers expression and promotes proteasomal degradation of Bcr-Abl and induces apoptosis of imatinib mesylate- sensitive or -refractory chronic myelogenous leukemia-blast crisis cells. Cancer Res. 2003, 63, 5126-5135.
37. Qian, D.Z.; Kato, Y.; Shabbeer, S.; Wei, Y.; Verheul, H.W.; Salumbides, B.; Sanni, T.; Atadja, P.; Pili1, R. Targeting tumor angiogenesis with histone deacetylase inhibitors: the hydroxamic acid derivative LBH589. Clin. Cancer Res. 2006, 12, 634-642.
38. Catley, L.; Weisberg, E.; Kiziltepe, T.; Tai, Y-T.; T Hideshima,.; Neri, P.; Tassone, P.; Atadja, P.; Chauhan, D.; Munshi, N. C.; Anderson, K. C..Aggresome induction by proteasome inhibitor bortezomib and α-tubulin hyperacetylation by tubulin deacetylase (TDAC) inhibitor LBH589 are synergistic in myeloma cells. Blood. 2006, 108, 3441-3449.
39. Prince, H.M.; Bishton. M. Panobinostat (LBH589):a novel pan-deacetylase inhibitor with activity in T cell lymphoma. Hematology Meeting Reports 2009, 3, 33-38.
40. Bertolini, G.; Biffi, M.; Leoni, F.; Mizrahi, J.; Pavich, G.; Mascagni, P. Compounds with anti-inflammatory and immunosuppressive activities. PCT Int. Appl. WO 6034096 (abstr.).
41. Leoni, F.; Fossati, G.; Lewis, C. E.; Lee, J-K.; Porro, G.; Pagani, P.; Modena, D. Moras, M. L.; Pozzi, P.; Reznikov, L. L.; Siegmund, B.; Fantuzzi, G.; Dinarello, A.C.; Mascagni, P. The Histone deacetylase inhibitor ITF2357 reduces production of pro-inflammatory cytokines in vitro and systemic inflammation in vivo. Mol. Med. 2005, 11, 1-15.
42. Armeanua, S.; Pathil, A.; Venturelli, S.; Mascagni, P.; Weiss, S. T.; Gottlicher, M.; Gregor, M.; Lauer, M. U.; Bitzer, M. Apoptosis on hepatoma cells but not on primary hepatocytes by histone deacetylase inhibitors valproate and ITF2357. J. Hepatol. 2005, 42, 2210-217.
43. Furumai, R.; Matsuyama, A.; Kobashi, N.; Lee, K.H.; Nishiyama, M.; Nakajima, H.; Tanaka, A.; Komatsu, Y.; Nishino, N.; Yoshida, M.; Horinouchi, S. FK228 (depsipeptide) as a natural prodrug that inhibits class I histone deacetylases. Cancer Res. 2002, 62, 4916-21.
44. Ueda, H.; Nakajima, H.; Hori, Y.; Fujita. T.; Nishimura, M.; Goto, T.; Okuhara, M. FR901228, a novel antitumor bicyclic depsipeptide produced by Chromobacterium violaceum No. 968. I. Taxonomy, fermentation, isolation, physico-chemical and biological properties, and antitumor activity. J. Antibiot. (Tokyo). 1994, 47, 301-310 (abstr.).
45. Zhang, Z.; Yang, Z.; Meanwell, A. N.; Kadow, F. J.; Wang, T. A general method for the preparation of 4- and 6-azaindoles. J. Org. Chem. 2002, 67, 2345-2347.
46. Li, J. J. et al. (2005). Name reactions in heterocyclic chemistry.
47. Huestis, P. M.; Fagnou , K. Site-selective azaindole arylation at the azine and azole rings via N-oxide activation. Org. Lett. 2009, 11, 1357-1360.
48. Mahboobi, S.; Sellmer, A,; Hocher, H.; Garhammer, C.; Pongratz, H.; Maier, T.; Ciossek, T.; Beckers, T. 2-Aroylindoles and 2-aroylbenzofurans with N-hydroxyacrylamide substructures as a novel series of rationally designed histone deacetylase inhibitors. J. Med. Chem. 2007, 50, 4405-4418.
49. Vinodkumar R.; Vaidya, S. D.; Kumar, B. V. S.; Bhise, U. N.; Bhirud, S. B.; Mashelkar, U. C. Synthesis, anti-bacterial, anti-asthmatic and anti-diabetic activities of novel N-substituted 2-(4-styrylphenyl)-1H- benzimidazole and N-substituted-3-[4-(1H-benzimidazole-2-yl)- phenyl]-acrylic acid tert-butyl ester. J. Polym. Sci. A. 2008, 14, 37-49.
50. Deng, X.; Mani, S. N. A facile, environmentally benign sulfonamide synthesis in water. Green Chem. 2006, 8, 835-838.

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系統識別號 U0007-0607201016194500
論文名稱(中文) 合成以蛇床子與肉桂酸為基礎之羥基醯胺作為組蛋白去乙醯酶抑制劑並促進神經母細胞瘤分化
論文名稱(英文) Synthesis of Osthole- and Cinnamate-Based Hydroxamates as Histone Deacetylase Inhibitors with Promoting Neuroblastoma Cell Differentiation
校院名稱 臺北醫學大學
系所名稱(中) 生藥學研究所
系所名稱(英) Graduate Institute of Pharmacognosy
學年度 98
學期 2
出版年 99
研究生(中文) 陽蘋
學號 M303097004
學位類別 碩士
語文別 中文
口試日期 2010-06-22
論文頁數 100頁
口試委員 指導教授-黃偉展
委員-張崇毅
委員-林俊茂
關鍵字(中) 組蛋白去乙醯酶
抑制劑、蛇床子、神經母細胞瘤
關鍵字(英) Histone deacetylase (HDAC) inhibitors, osthole, neuroblastoma
學科別分類
中文摘要 組蛋白去乙醯酶抑制劑為目前標靶治療的研究之發展趨勢,分析組蛋白去乙醯酶抑制劑的化學結構分為三個部分分別為:疏水性的基團、疏水性的鏈長、與鋅螯合產生活性的基團,本論文主要合成(1)以蛇床子為基礎之氮-羥基直鏈醯胺(2)芳香環取代之羥基肉桂酸醯胺,以作為有效及選擇性的組蛋白去乙醯酶抑制劑(HDACi),並具有促進神經細胞瘤分化作用。
我們以氫化後的蛇床子素為模板合成不同碳鏈長度之氮-羥基直鏈醯胺6a~6k,並測試其抑制子宮頸癌細胞之組蛋白去乙醯酶(HeLa cell nuclear extract HDAC)之活性。其中化合物6c、6d、6g、6k (IC50=24.6, 28.9, 22.2, 23.8 nM)比suberoylanilide hydroxamic acid (SAHA, IC50=41.7 nM)效果更強,進一步測試組蛋白去乙醯酶亞型(HDAC-1, -4, -6, -8)活性,在第一、四、六型組蛋白去乙醯酶 (HDAC-1, -4, -6)抑制活性中,6c、6d、6g、6k均顯示與SAHA相當的活性,而在第八型組蛋白去乙醯酶 (HDAC8)的酵素抑制活性中6c、6d、6g、6k顯示比SAHA較強之活性,尤其6c為SAHA 12倍。近來有研究指出第八型組蛋白去乙醯酶與神經母細胞瘤分化(neuroblastoma cell differentiation)有關,因此我們將化合物6c、6d、6g、6k進行神經母細胞瘤SH-SY5Y 細胞活性實驗,發現6c比SAHA具有較佳之促進神經母細胞瘤分化的效果,進而將6c與SAHA分別與第八型組蛋白去乙醯酶進行分子模擬分析(molecular modeling analysis),顯示6c與SAHA共同擁有之苯環都是作用酵素疏水區的口袋表面,但不同的是,與SAHA相較,由於6c苯環上較SAHA多了側鏈dimemthylpropane及 N-hydroxypropamide,故可與HDAC8產生更多的疏水作用 (hydrophobic interaction),推測因此6c較SAHA具有更好的抑制活性。
以氮-羥基肉桂酸醯胺為模板,在鄰位與對位分別接上不同取代基之苯甲基,共合成十四個化合物11a~11e、17a~17f、22、27、32進行第一,四,六,八型組蛋白去乙醯酶(HDAC1, -4, -6, -8)抑制活性測定。在第八型(HDAC8)抑制活性,化合物11a、11c、11d、22、32較SAHA效果強,第一,六型(HDAC1, -6)均比SAHA弱,在第四型(HDAC4)則與SAHA類似,較不具抑制性,此結果顯示化合物11a、11c、11d、22、32為具有選擇性之第八型組蛋白去乙醯酶抑制劑(HDAC 8 inhibitor), 而在神經母細胞瘤SH-SY5Y 細胞活性實驗發現,22與32均較SAHA具明顯促進神經母細胞瘤分化的效果。
綜合以上的活性試驗的結果顯示,以蛇床子為基礎之氮-羥基直鏈醯胺系列化合物,其對組蛋白去乙醯酶亞型(HDAC-1, -4, -6, -8)抑制活性均較SAHA強或與SAHA相當,此類化合物屬於為pan-histone deacetylase (HDAC) inhibitors。而芳香環取代之羥基肉桂酸醯胺系列化合物,雖對組蛋白去乙醯酶亞型(HDAC-1, -6)抑制活性遠比SAHA來得小,但對HDAC-8有很好的抑制活性,具有成為治療神經母細胞瘤藥物之潛力。

英文摘要 Histone deacetylase (HDAC) inhibitors had been used as potential agents for targeted cancer chemotherapy. From previous publications, HDAC inhibitors were know to consist of three major parts: a hydrophobic cap for surface recognition, a zinc-chelating group and a hydrophobic linker between the two functional groups. We focus on the synthesis of osthole- and cinnamate-based hydroxamates as histone deacetylase inhibitors and test their neuronal differentiation activities on neuroblastoma cells (SH-SY5Y).
Eleven novel osthole-based N-hydroxamates, compound 6a~6k, were synthesized and screened for HDAC inhibitory activity by using HeLa nuclear extract. In this screening, compounds 6c, 6d, 6g and 6k showed similar activity (the IC50 was 24.6, 28.9, 22.2 and 23.8 nM, respectively) as suberoylanilide hydroxamic acid (SAHA, the IC50 was 41.7 nM), the potent HDAC inhibitor for the treatment of cutaneous T-cell lymphoma (CTCL) approved by FDA in 2006. After screening on different classes of HDAC enzymes, our compounds were active against both class I (HDAC-1, -8) and class II (HDAC-4, -6) indicated that all of them showed similar inhibitory activity against HDAC-1, -4 and -6, but were much more active against HDAC-8, especially the compound 6c with 12-fold active than SAHA. In the docking analysis, the branched side chains, the dimthylpropane and N-hydroxypropamide groups, of compound 6c made the hydrophobic interaction on HDAC 8 stronger than that of SAHA. Furthermore, the compound 6c also showed the best cellular activity on the promotion of neurite outgrowth and neuronal differentiation on SH-SY5Y neuroblastoma cells in these series of analogues.
Fourteen novel N-hydroxycinnamides, comuond 11a~11e, 17a~17f, 22, 27 and 32, substituted with ortho- or para- benzyl derivatives were synthesized and screened for their HDAC inhibitory activity (HDAC1, -4, -6, -8). Compounds 11a, 11c, 11d, 22 and 32 were highly selective against HDAC-8 and were 9-, 3-, 5-, 15- and 12-fold, respectively potent than that of SAHA. But in neuroblastoma differentiation experiment, only compounds 22 and 32 showed significant promoting effect, others lost their activity in the cell model system.
By these results, we found that osthole-based hydroxamates exhibited SAHA-like activity against HDAC-1, -4, -6 and -8. and were likely a wonderful skeleton for pan HDAC inhibitor design. On the other hand, the benzyl substituted N-hydroxycinnamides were selective toward HDAC 8 over other subtypes (HDAC-1, -4, -6). We think that this skeleton might be a good lead for the design of novel HDAC inhibitor with HDAC-8 selectivity. Although the preliminary data showed that some of the compounds were effective on the promotion of the neuroblastoma differentiation, but there still further estimations needed to be done to determinate their therapeutic potential on neuroblastoma.
論文目次 目錄
中文摘要 i
Abstract iii
表目錄 viii
流程圖目錄 ix
圖目錄 x
附圖目錄 xi
一、緒論及研究目的 - 1 -
二、結果與討論 - 14 -
1. 化學合成 - 14 -
1.1以蛇床子為基礎之氮-羥基直鏈醯胺之合成 - 14 -
1.2 芳香環取代之羥基肉桂酸醯胺之合成 - 18 -
2. 化合物(6a~6k)對於抑制子宮頸癌細胞核萃取之組蛋白去乙醯酶(HeLa cell nuclear extract histone deacetylase)之活性 - 23 -
3. 化合物6c、6d、6g、6k、11a、11c、11d、22 、32 對組蛋白去乙醯酶1、4、6、8型(HDAC1,-4,-6,-8)酵素抑制活性 - 24 -
4. 以西方墨點法偵測化合物 6c、 6g、 6k、 22、 32影響 SH-SY5Y細胞株神經分化標記之表現: - 27 -
5. 化合物6c、32與SAHA對HDAC 8之分子模擬(molecular modeling)- 30 -
三、結論 - 32 -
四、實驗方法 - 33 -
1. 儀器與材料 - 33 -
(1). 一般儀器及方法 - 33 -
(2). 試劑及溶劑來源 - 34 -
2. 化學合成步驟及物理資料 - 37 -
3. 生物活性分析 - 88 -
(1). HDAC activity assay - 88 -
(2). Compound-Induced Neuronal Differentiation of Neuroblastoma Cells……………………………………………………………………...-89-
(3).Western Blotting - 90 -
(4). Molecular modeling - 91 -
五、參考文獻(References) - 93 -
六、附圖 - 95 -


參考文獻 1. H. F. Tien, Molecular Therapy in Hematologic Malignancies. Formosan. J. Med 7, 212-221 (2003).
2. R. K. Hsieh, Molecular Targeted Therapy for Solid Tumors. Formosan. J. Med 7, 222-226 (2003).
3. C. Hildmann, D. Wegener, D. Riester, R. Hempel, A. Schober, J. Merana , L. Giurato,S. Guccione, T. K. Nielsen, R. Ficner, A. Schwienhorst, Substrate and inhibitor specificity of class 1 and class 2 histone deacetylases. J. biotechnology 124, 258-270 (2006).
4. Y. Kawaguchi, J. J. Kovacs, A. McLaurin, J. M. Vance, A. Ito, T. Yao, The deacetylase HDAC6 regulates aggresome formation and cell viability in response to misfolded protein stress. Cell 115, 727-738 (2003).
5. C. Hildmann, D. Riester, A. Schwienhorst, Histone deacetylases—an important class of cellular regulators with a variety of functions. Appl. Microbiol. Biotechnol. 75, 487-489 (2007).
6. B. W. Dymock, H. Wang, New patented histone deacetylase inhibitors. Expert Opin. Ther. Patents 19, 1727-1757 (2009).
7. G. Elaut, V. Rogiers, T. Vanhaeckt, The Pharmaceutical Potential of Histone Deacetylase Inhibitors. Curr. Pharm. Des.13, 2584-2620 (2007).
8. J. E. Bolden, M. J. Peart, R. W. Johnstone, Anticancer activities of histone deacetylase inhibitors. Nat. Rev. 5, 769-784 (2006).
9. M. Paris, M. Porcelloni, M. Binaschi, D. Fattori. Histone Deacetylase Inhibitors: From Bench to Clinic. J. Med. Chem. 51, 1505-1529 (2008).
10. V. R. Richon, V. M. Richon. Mechanisms of Resistance to Histone Deacetylase Inhibitors and Their Therapeutic Implications. Clin. Cancer. Res. 13, 7237-7242 (2007).
11. W. S. Xu, R. B. Parmigiani, P. A. Marks. Histone deacetylase inhibitors: molecular mechanisms of action. Oncogene 26, 5541-5552 (2007).
12. S. Chen, J. Ye, I, Kijima , D. Evans, The HDAC inhibitor LBH589 (panobinostat) is an inhibitory modulator of aromatase gene expression. Proc. Natl. Acad. Sci. 107, 11032-11037 (2010).
13. S. Grant, Vorinostat. Nat. Rev. 6, 21-22 (2007).
14. S. A. Kavanaugh, L. A. White, J. M. Kolesar. Vorinostat: A novel therapy for the treatment of cutaneous T-cell lymphoma. Am. J. Health. Syst. Pharm. 67, 793-797 (2010).
15. J. Abraham, Vorinostat in cutaneous T-cell lymphoma. Commun. Oncology 4, 384-386 (2007).
16. D. R. Davie, Covalent modifications of histones: expression from chromatin templates. Curr. Opin. Genet. Dev. 8, 173-178 (1998).
17. W. D.Cress, E. Seto, Histone Deacetylases, Transcriptional Control, and Cancer. J. Cell. Physiol . 184, 1-16 (2000).
18. A. J. Ruijter, A. H. van Gennip, H. N. Caron, S. Kemp, A. B. van Kuilenburg,
Histone deacetylases (HDACs): characterization of the classical HDAC family. J. Biol. chem. 370, 737-749 (2003).
19. P. Marks, R. A. Rifkind, V. M. Richon, Breslow, T. Miller, W. K. Kelly, Histone deacetylases and cancer: causes and therapies. Nat. Rev. Cancer. 1, 194-202 (2001).
20. P. A. Wade, Transcriptional control at regulatory checkpoints by histone deacetylases:molecular connections between cancer and chromatin. Hum. Mol. Genet. 10, 693-698 (2001).
21. D. M. C. Vigushin, R. Charles. Histone deacetylase inhibitors in cancer treatment. . Anticancer Drugs 13, 1-13 (2002).
22. B. J. North, B. L. Marshall, M. T. Borra, J. M. Denu, E. Verdin. The human Sir2 ortholog, SIRT2, is an NAD+-dependent tubulin deacetylase. Mol. Cell 11, 437-444 (2003).
23. O. Witt , H. E. Deubzer, T. Milde, I. Oehme. HDAC family: What are the cancer relevant targets? Cancer Lett. 277, 8-21 (2009).
24. K. B. Glaser, J. Li, M. J. Staver, R. Q. Wei, D. H. Albert, S. K. Davidsen, Role
of class I and Class II histone deacetylases in carcinoma cells using siRNA. Biochem. Biophys. Res. Commun. 310, 529-536 (2003).
25. P. Gallinari, S. D. Marco, P. Jones, M. Pallaoro, C. Steinkühler, HDACs, histone deacetylation and gene transcription: from molecular biology to cancer therapeutics. Cell Res. 17, 195-211 (2007).
26. S. F. Sleiman, M. Basso, L. Mahishi, A. P. Kozikowski, M. E. Donohoe, B. Langley, R. R. Ratan, Putting the ‘HAT’ back on survival signalling: the promises and challenges of HDAC inhibition in the treatment of neurological conditions. Expert Opin. Investig. Drugs 573-584 (2009).
27. M. Kilgore, C. A. Miller, D. M. Fass, K. M. Hennig, S. J. Haggarty, J. D. Sweatt, G. Rumbaugh, Inhibitors of Class 1 Histone Deacetylases Reverse Contextual Memory Deficits in a Mouse Model of Alzheimer’s Disease. Neuropsychopharmacology 35, 870-880 (2010).
28. A. Mogal, A. S. Abdulkadir, Effects of Histone Deacetylase Inhibitor (HDACi); Trichostatin-A (TSA) on the expression of housekeeping genes. Mol. Cell. Probes. 20, 81-86 (2006).
29. A. Monks, C. D. Hose, Pezzoli, S. , Kondapaka, G. Vansant, K. D. Petersen, M. Sehested, J. Monforte, R. H. Shoemaker, Gene expression-signature of belinostat in cell lines is specific for histone deacetylase inhibitor treatment,
with a corresponding signature in xenografts. Anticancer Drugs 20, 682-692 (2009).
30. H. Wang, N. Yu, H. Song, D. Chen, Y. Zou, W. Deng, P. L. Lye, J. Chang, M. Ng, E. T. Sun, K. Sangthongpitag, X. Wang, X. Wu, H. H. Khng, L. Fang, S. K. Goh, W. C. Ong, Z. Bonday, W. Stunkel, A. Poulsen, M. Entzeroth, N-Hydroxy-1,2-disubstituted-1H-benzimidazol-5-yl acrylamides as novel
histone deacetylase inhibitors: Design, synthesis, SAR studies, and in vivo antitumor activity. Bioorg. Med. Chem. Lett. 19, 1403-1408 (2009).
31. S. Okabe, T. Tauchi, A. Nakajima, G. Sashida, A. Gotoh, H. E. Broxmeyer, J. H. Ohyashiki, K. Ohyashiki, Depsipeptide (FK228) preferentially induces apoptosis in BCR/ABL-expressing cell lines and cells from patients with chronic myelogenous leukemia in blast crisis. Stem. Cells. Dev. . 16, 503-514 (2007).
32. G. Garcia-Manero, H. Yang, C. Bueso-Ramos, A. Ferrajoli, J. Cortes, W. G. Wierda, S. Faderl, C. Koller, G. Morris, G. Rosner, A. Loboda, V. R. Fantin, S. S. Randolph, J. S. Hardwick, J. F. Reilly, C. Chen, J. L. Ricker, J. P. Secrist, V. M. Richon, S. R. Frankel, H. M. Kantarjian, Phase 1 study of the histone deacetylase inhibitor vorinostat (suberoylanilide hydroxamic acid [SAHA]) in patients with advanced leukemias and myelodysplastic syndromes. Blood. 111, 1060-1066 (2007).
33. M. T. Buckley, J. Yoon, H. Yee, L. Chiriboga, L. Liebes, G, Ara, X. Qian, D. F. Bajorin, T. T. Sun, X. R. Wu, I. Osman, The histone deacetylase inhibitor belinostat (PXD101) suppresses bladder cancer cell growth in vitro and in vivo. J. Transl. Med. 5, 1479-1587 (2007).
34. Q. C. Ryan, D. Headlee, M. Acharya, A. Sparreboom, J. B. Trepel, J. Ye, W. D. Figg, K. Hwang, E. J. Chung, A. Murgo, G.i Melillo, Y. Elsayed, M. Monga, M. Kalnitskiy, J. Zwiebel, E. A. Sausville, Phase I and Pharmacokinetic Study of MS-275, a Histone Deacetylase Inhibitor, in Patients With Advanced and Refractory Solid Tumors or Lymphoma. J. Cli .Oncol. 23, 3912-3922 (2005).
35. S. Minucci, P. G. Pelicci, Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer. Nat. Rev. 6, 39-51 (2006).
36. Q. Lu, Y. T. Yang, C. S. Chen, M. Davis, J. C. Byrd, M. R. Etherton, A. Umar, C. S. Chen, Zn2+-Chelating Motif-Tethered Short-Chain Fatty Acids as a Novel Class of Histone Deacetylase Inhibitors. J. Med. Chem. 47, 467-474 (2004).
37. G. Dasmahapatra, D. Lembersky, L. Kramer, R. I. Fisher, J. Friedberg, P. Dent, S. Grant, The pan-HDAC inhibitor vorinostat potentiates the activity of the proteasome inhibitor carfilzomib in human DLBCL cells in vitro and in vivo. Blood. 115, 4478-4487 (2010).
38. O. Witt, H. E. Deubzer, M. Lodrini, T. Milde, I. Oehme, Targeting Histone Deacetylases in Neuroblastoma. Curr. Pharm. Design 15, 436-447 (2009).
39. S. Chen, G.C. Owens, H. Makarenkova, D. B. Edelman, HDAC6 regulates mitochondrial transport in hippocampal neurons. PLoS One. 5, 1-11 (2010).
40. I. Oehme, H. E. Deubzer, D Wegener, D. Pickert, J. P. Linke, B. Hero, A. Kopp-Schneider, F. Westermann, S. M. Ulrich, A. von Deimling, M. Fischer, O. Witt, Histone Deacetylase 8 in NeuroblastomaTumorigenesis. Clin Cancer Res. 15, 91-99 (2009).
41. G. M. Brodeur, Neuroblastoma: Biological insights into a clinical enigma. Nat.Rev. Cancer 3, 203-216 (2003).
42. L. You, S. Feng, R. An, X. Wang, Osthole: a promising lead compound for drug discovery from a traditional Chinese medicine (TCM). Nat Prod Commun 4, 297-302 (2009).
43. T. Fujioka, K. Furumi, H. Fujii, H. Okabe, K. Mihashi, Y. Nakano, H. Matsunaga, M. Katano, M. Mori, Antiproliferative constituents from umbelliferae plants. V. A new furanocoumarin and falcarindiol furanocoumarin ethers from the root of Angelica japonica. Chem. Pharm. Bull. 47, 96-100 (1999).
44. P. L. Kuo, Y. L. Hsu, C. H. Chang, J. K. Chang, Osthole-Mediated Cell Differentiation through Bone Morphogenetic Protein-2/p38 and Extracellular Signal-Regulated Kinase 1/2 Pathway in Human Osteoblast Cells. Pharmacol. Exp. Ther. 314, 1290-1299 (2005).
45. W. J. Huang, C. C. Chen, S. W. Chao, S. S. Lee, F. L. Hsu, Y. L. Lu , M. F. Hung, C. I. Chang, Synthesis of N-Hydroxycinnamides Capped with a Naturally Occurring Moiety as Inhibitors of Histone Deacetylase. Chem. Med. Chem. 5, 598-607 (2010).
46. K. KrennHrubec, B. L. Marshall, M. Hedglin, E. Verdin, S. M. Ulrich. Design and evaluation of ‘Linkerless’ hydroxamic acids as selective HDAC8 inhibitors. Bioorg. Med. Chem. Lett.17, 2874–2878 (2007).
47. G. M. Morris, R. Huey, W. Lindstrom, M. F. Sanner, R. K. Belew, D. S. Goodsell, A. AutoDock4 and AutoDockTools4: Automated Docking with Selective Receptor FlexibilityJ. Oloson, J. Comput. Chem. 30, 2785-2791(2009).


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系統識別號 U0007-0807201016173200
論文名稱(中文) 以天然物為cap與氮-羥基肉桂酸醯胺結合作為組蛋白去乙醯酶抑制劑
論文名稱(英文) Synthesis of N-Hydroxycinnamides Capped with a Naturally Occurring Moiety as Inhibitors of Histone Deacetylase
校院名稱 臺北醫學大學
系所名稱(中) 生藥學研究所
系所名稱(英) Graduate Institute of Pharmacognosy
學年度 98
學期 2
出版年 99
研究生(中文) 趙世偉
學號 M303097001
學位類別 碩士
語文別 中文
口試日期 2010-06-22
論文頁數 137頁
口試委員 指導教授-黃偉展
委員-陳彥州
委員-張崇毅
關鍵字(中) 組蛋白
組蛋白去乙醯酶
關鍵字(英) Histone
HDAC
學科別分類
中文摘要 研究顯示,組蛋白去乙醯酶抑制劑可望發展成為抗癌藥物,所有已知之組蛋白去乙醯酶抑制劑 (HDACi),從化學結構上包含了三個藥理活性基團:與鋅產生螯合之基團,疏水性鏈長,與疏水性的辨認部位。在本研究我們以具有疏水性之中藥成份蛇床子素 (osthole),來當作疏水性的辨認部位,合成一系列以N-hydroxycinnamides為基本架構之組蛋白去乙醯酶抑制劑 (HDACi),以探討蛇床子素 (osthole) 對於活性的效果。我們合成了九個結合蛇床子素 (osthole) 之N-hydroxacinnamides (9a~9i),並同時進行酵素抑制活性測試,發現化合物9d, 9e, 9g 具有顯著抑制子宮頸癌細胞萃取之組蛋白去乙醯酶 (HeLa cell nuclear extract HDAC) (IC50: 24.5, 20.0, 19.6 nM),其抑制活性相當於目前臨床上用於治療表皮T細胞淋胞癌 (cutaneous T-cell lymphoma, CTCL) 之組蛋白去乙醯酶抑制劑 (HDACi) suberoylanilide hydroxamic acid (SAHA, IC50: 24.5 nM),另外在組蛋白去乙醯酶1型 (HDAC1) 和組蛋白去乙醯酶6型 (HDAC6) 之酵素抑制活性上,化合物9d及9e具有和SAHA類似之活性,9g則對組蛋白去乙醯酶抑制劑1型 (HDAC1) 具有較好的選擇性,從乙醯化組蛋白3型 (acetylated H3) 與乙醯化α-微管蛋白 (acetylated α-tubulin) 之促進實驗中,化合物9d, 9e, 9g與SAHA相比較,9d有明顯之促進乙醯化之作用,顯示其具有好之細胞效果,接著將9d與SAHA進行分子結合模擬分析,顯示9d之蛇床子素 (osthole) 基團與SAHA的苯環都是作用酵素口袋表面疏水區,且蛇床子素 (osthole) 之側鏈經過修飾,可能產生對第Ⅱa型組蛋白去乙醯酶 (class Ⅱa HDAC) 選擇性抑制,以上這些結果證實,以蛇床子素 (osthole) 為疏水性cap與N-hydroxacinnamides相結合,可發展出有效之組蛋白去乙醯酶抑制劑 (HDACi)。
英文摘要 Histone deacetylase (HDAC) inhibitors are regarded as promising therapeutics for the treatment of cancer. All reported HDAC inhibitors contain three pharmacophoric features: a zinc-chelating group, a hydrophobic linker, and a hydrophobic cap for surface recognition. In this study we investigated the effectiveness of osthole, a hydrophobic Chinese herbal compound, as the surface recognition cap in hydroxamate-based compounds as inhibitors of HDAC. Nine novel osthole-based N-hydroxycinnamides were synthesized and screened for enzyme inhibition activity. Compounds 9d, 9e, 9g exhibited inhibitory activities (IC50=24.5, 20.0, 19.6 nM) against nuclear HDACs in HeLa cells comparable to that of suberoylanilide hydroxamic acid (SAHA; IC50=24.5 nM), a potent inhibitor clinically used for the treatment of cutaneous T-cell lymphoma (CTCL). While compounds 9d and 9e showed SAHA-like activity towards HDAC1 and HDAC6, compound 9g was more selective for HDAC1. Compound 9d exhibited the best cellular effect, which was comparable to that of SAHA, of enhancing acetylation of either α-tubulin or histone H3. Molecular docking analysis showed that the osthole moiety of compound 9d may interact with the same hydrophobic surface pocket exploited by SAHA and it may be modified to provide class-specific selectivity. These results suggest that osthole is an effective hydrophobic cap when incorporated into N-hydroxycinnamide-derived HDAC inhibitors.
論文目次 中文摘要 i
Abstract iii
表目錄 vii
流程圖目錄 viii
圖目錄 ix
一、緒論及研究目的 - 1 -
二、結果與討論 - 9 -
1. 化學合成 - 9 -
2. 化合物 (9a-i) 對於抑制子宮頸癌細胞萃取之組蛋白去乙醯酶(HeLa cell nuclear extract histone deacetylase) 與抑制肝癌細胞 (HepG2) 生長之活性 - 12 -
3. 化合物9d, 9e, 9g, 9i對組蛋白去乙醯酶1、6型 (HDAC1,-6)
酵素抑制活性 - 14 -
4. 化合物9d, 9e, 9g對於肝癌細胞HepG2α-微管蛋白乙醯化
(acetylation α-tubulin) 及組蛋白三型乙醯化 (acetylation
histone 3) 之影響 - 15 -
5. 化合物9d之分子模擬(molecular modeling) - 17 -
6. 化合物 9d 對於正常細胞之細胞毒性 - 19 -
三、結論 - 20 -
四、實驗部份 - 21 -
I. 儀器與實驗材料 - 21 -
1. 一般儀器及方法 - 21 -
II. 化學合成步驟及物理資料 - 24 -
III. 生物活性測試方法 - 49 -
1. HDAC activity assay - 49 -
2. The sulforhodamine B (SRB) growth inhibition assay - 50 -
3. Western blot analysis - 51 -
五、參考文獻(References) - 52 -
六、附圖 - 60 -
參考文獻 1.Matsudaira, P.T.L.; Harvey, F; Arnold, B.; Kaiser, C.;
Monty, K.; Matthew, P.; Anthony, B.; Hidde, P., Molecular
Cell Biology, 2007.
2.Alberts, B., Molecular biology of the Cell. 2002, New
York.
3.Jemal, A.; Murray, T.; Ward, E.; Samuels, A.; Tiwari,
R.C.; Ghafoor, A.; Feuer, E.J.; Thun, M.J., CA Cancer J.
Clin., 2005, 55, 10.
4.Harper, D.M.; Franco, E.L.; Wheeler, C.; Ferris, D.G.;
Jenkins, D.; Schuind, A.; Zahaf, T.; Innis, B.; Naud, P.;
De Carvalho, N.S.; Roteli-Martins, C.M.; Teixeira, J.;
Blatter, M.M.; Korn, A.P.; Quint, W.; Dubin, G., Lancet,
2004, 364, 1757.
5.David, M.K.; Anna, C., Nature Reviews Microbiology, 2008,
6, 211.
6.Luger, K.; Mader, A.W.; Richmond, R.K.; Sargent, D.F.;
Richmond, T.J., Nature, 1997, 389, 251.
7.Davie, J.R., Curr Opin Genet Dev, 1998. 8, 173.
8.Cress, W.D. ; Seto, E.; J Cell Physiol, 2000, 184, 1.
9.Munshi, A.; Shafi, G.; Aliya. N.; Jyothy A., J. Genet.
Genomics, 2009, 36, 75.
10.Takayoshi, S.; Naoki, M., Curr. Med. Chem., 2006, 13,
935.
11.Herman, J.G.; Baylin, S.B., New. Engl. J. Med., 2004,
350, 947.
12.Roth, S.Y.; Denu, J.M.; Allis, C.D., Annu. Rev.
Biochem., 2001, 70, 81.
13.David, E.S.; Shelley, L.B., Microbiol. Mol. Biol. Rev.,
2000, 64, 435.
14.Hassig, C.A.; Schreiber, S.L., Curr. Opin. Chem. Biol,
1997, 1, 300.
15.Gregoire, S.; Yang, X.J., Mol. Cell. Biol., 2005, 25,
2873.
16.Khochbin, S.; Verdel, A.; Lemercier, C.; Seigneurin-
Berny, D., Curr. Opin. Genet. Dev., 2001, 11, 162.
17.Annemieke, J.M.; Ruijter, D.; Albert, H.; GENNIP, V.;
CARON, H.N.; Kemp, S.; Andre, B.P.; Kuilenburg V.,
Biochem. J., 2003, 370, 737.
18.Gregoretti, I.V.; Lee, Y.M.; Goodson, H.V., J. Mol.
Biol, 2004, 338, 17.
19.Robertson, K.D.; Wolffe, A.P., Nature Rev. Genet., 2000,
1, 11.
20.Feng, Q.; Zhang, Y., Genes & Dev., 2001, 15, 827.
21.Fuks, F.B.; Wendy, A.; Godin, N.; Kasai, M.; Kouzarides,
T., EMBO J., 2001, 20, 2536.
22.Verdin, E.; Dequiedt, F.; Kasler, H.G., Trends Genet.,
2003, 19, 286.
23.Mckinsey, T.A.; Zhang, C.L.; Lu, J.; Olson, E. N.,
Nature, 2000, 408, 106.
24.Wade, P.A., Hum Mol Genet, 2001, 10, 693.
25.Hubbert, C.; Guardiola, A.; Shao, R.; Kawaguchi, Y.;
Ito, A.; Nixon, A.; Yoshida, M.; Wang, X.F.; Yao, T.P.,
Nature, 2002, 417, 455.
26.North, B.J.; Marshall, B.L.; Borra, M.T.; Denu, J.M.;
Verdin, E., Mol. Cell, 2003, 11, 437.
27.Vigushin, D.M.; Coombes, R.C., Anti-Cancer Drugs, 2002,
13, 1.
28.Olaf, W. ; Hedwig, E.D.; Till, M.; Ina, O., Cancer
Lett., 2009, 277, 8.
29.Abraham, J., Community Oncology, 2007, 4, 384.
30.Glaser, K.B.; Li, J.; Staver, M.J.; Wei, R.Q.; Albert,
D.H.; Davidsen, S.K., Biochem Biophys Res Commun, 2003,
310, 529.
31.Paris, M.; Porcelloni, M.; Binaschi, M.; Fattori, D., J
Med Chem, 2008, 51, 1505.
32.Yoshida, M.; Kijima, M.; Akita, M.; Beppu, T., J Biol
Chem, 1990, 265, 17174.
33.Han, J.W.; Ahn, S.H.; Park, S.H.; Wang, S.Y.; Bae, G.U.;
Seo, D.W.; Kwon, H. K.; Hong, S.; Lee, H.Y.; Lee, Y.W.;
Lee, H.W., Cancer Res, 2000, 60, 6068.
34.Yoshida, M.; Horinouchi, S.; Beppu, T., BioEssays, 1995,
17, 423.
35.Closse, A.; H. R., Helv. Chim. Acta, 1974, 57, 553.
36.Kawai, M.; Rich, D.H.; Walton, J.D., Biochem. Biophys.
Res. Comm., 1983, 111, 398.
37.Itazaki, H.; Nagashima, K.; Sugita, K.; Yoshida, H.;
Kawamura, Y.; Yasuda, Y.; Matsumoto, K.; Ishii, K.;
Uotani, N.; Nakai, H.; Terui, A.; Yoshimatsu, S.;
Ikenishi, Y.; Nakagawa, Y., J. Antibiot., 1990, 63, 1524.
38.Furumai, R.; Matsuyama, A.; Kobashi, N.; Lee, K.H.;
Nishiyama, M.; Nakajima, H.; Nakajima, A.; Komatsu, Y.;
Nishino, N.; Yoshida, M.; Horinouchi, S., Cancer Res.,
2002. 62, 4916.
39.Fournel, M.; Trachy-Bourget, M.C.; Theresa Y.P.; Kalita,
A., Cancer Res., 2002, 62, 4325.
40.Richon, V.M.; Webb, Y.; Merger, R.; Sheppard, T.;
Jursic, B.; Ngo, L.; Civoli, F.; Breslow, R.; Rifkind,
R.A.; Marks, P.A., Proc. Natl. Acad. Sci. U.S.A., 1996,
93, 5705.
41.Richon, V.M.; Emiliani, S.; Verdin, E.; Webb, Y.;
Breslow, R.; Rifkind R.A.; Paul, A.M., Proc. Natl. Acad.
Sci. U.S.A., 1998, 95, 3003.
42.Cohen, L.A.; Amin, S.; Marks, P.A.; Rifkind, R.A.;
Desai, D.; Richon, V.M., Anticancer Res., 1999, 19, 4999.
43.Wittich, S.; Scherf, H.; Xie, C.; Brosch, G.; Loidl, P.;
Gerhauser, C.; Jung, M., J. Med. Chem., 2002, 45, 3296.
44.Furumai, R.; Komatsu, Y.; Nishino, N.; Khochbin, S.;
Yoshida, M.; Horinouchi, S., Proc. Natl. Acad. Sci.
U.S.A., 2001, 98, 87.
45.Komatsu, Y.; Tomizaki, K.Y.; Tsukamoto, M.; Kato, T.;
Nishino, N.; Sato, S.; Sato, T.; Tsuruo, T.; Furumai,
R.; Yoshida, M.; Horinouchi, S.; Hayashi, H., Cancer
Res., 2001, 61, 4459.
46.Plumb, J.A.; Finn, P.W.; Williams, R.J.; Bandara, M.J.;
Romero, M.R.; Watkins, C.J.; Thangue, N.B.; Brown, R.,
Mol. Cancer. Ther., 2003, 2, 721.
47.Remiszewski, S.W., Curr. Med. Chem., 2003, 10, 2393.
48.Remiszewski, S.W.; Sambucetti, L.C.; Bair, K.W.;
Bontempo, J., J. Med. Chem., 2003, 46, 4609.
49.Atadja, P.; Gao, L.; Kwon, P.; Trogani, N.; Walker, H.;
Hsu, M., Cancer Res., 2004, 64, 689.
50.Bouchain, G.; Leit, S.; Frechette, S.; Khalil, E.A.;
Lavoie, R., J. Med. Chem., 2003, 46, 820.
51.Besterman, J.M., Pacifichem, 2005.
52.Ueda, H.; Nakajima, H.; Hori, Y.; Fujita, T.; Nishimura,
M.; Goto, T.; Okuhara, M., J Antibiot., 1994, 47, 301.
53.Richon, V.M.; Emiliani, S.; Verdin, E.; Webb, Y.;
Breslow, R.; Rifkind, R.A.; Marks, P. A., Proc Natl Acad
Sci U S A 1998, 95, 3003.
54.Monks, A.; Hose, C.D.; Pezzoli, P.; Kondapaka, S.;
Vansant, G.; Petersen, K. D.; Sehested, M.; Monforte,
J.; Shoemaker, R.H., Anticancer Drugs, 2009, 20, 682.
55.Wang, H.; Yu, N.; Song, H.; Chen, D.; Zou, Y.; Deng, W.;
Lye, P.L.; Chang, J.; Ng, M.; Sun, E.T.; Sangthongpitag,
K.; Wang, X.; Wu, X.; Khng, H.H.; Fang, L.; Goh, S.K.;
Ong, W.C.; Bonday, Z.; Stunkel, W.; Poulsen, A.;
Entzeroth, M., Bioorg. Med. Chem. Lett., 2009, 19, 1403.
56.Bottomley, M.J.; Lo, S. P.; Di Giovine, P.; Cirillo, A.;
Scarpelli, R.; Ferrigno, F.; Jones, P.; Neddermann, P.;
De Francesco, R.; Steinkuhler, C.; Gallinari, P.; Carfi,
A., J. Biol. Chem., 2008, 283, 26694.
57.Nielsen, T.K.; Hildmann, C.; Dickmanns, A.;
Schwienhorst, A.; Ficner, R., J. Mol. Biol, 2005, 354,
107.
58.Taunton, J.; Hassig, C.A.; Schreiber, S.L., Science,
1996, 272, 408.
59.Vaisburg, A.; Bernstein, N.; Frechette, S.; Allan, M.;
Abou-Khalil E., Bioorg. Med. Chem. Lett., 2004, 14, 283.
60.Bouchain, G.; Delorme, D., Curr. Med. Chem., 2003, 10,
2359.
61.Woo, S.H.; Frechette, S.; Khalil, E.A.; Bouchain, G.;
Vaisburg, A.; Moradei, O.; Bernstein, N.; Leit, S.;
Allan, M.; Fournel, M.; Trachy-Bourget, Z.; Besterman,
J. M.; Delorme, D., J. Med. Chem., 2002, 45, 2877.
62.Frey, R.R.; Wada, C.K.; Garland, R.B.; Curtin, M.L.;
Michaelides, R.L.; Michaelides, J.; Pease, L.J.; Glaser,
K.B.; Marcotte, P.A.; Bouska,J.J.; Murphy, S.S.;
Davidsen, S.K., Bioorg. Med. Chem. Lett., 2002, 12, 3443.
63.Finnin, M.S.; Donigian, J.R.; Cohen, A.; Richon, V.M.;
Rifkind, R.A.; Marks, P.A.; Pavletich, N. P., Nature,
1999, 401, 188.
64.Somoza, J.R.; Skene, R.J.; Katz, B.A.; Mol, C.; Ho,
J.D., Structure, 2004, 12, 1325.
65.Vannini, A.; Volpari, C.; Filocamo, G.; Casavola, E. C.;
Brunetti, M.; Renzoni, D.; Chakravarty, P.; Paolini, C.;
De Francesco, R.; Gallinari, P.; Steinkuhler, C.; Di
Marco, S., Proc. Natl. Acad. Sci., 2004, 101, 15064.
66.Vannini, A.; Volpari, C.; Filocamo, G.; Casavola, E.C.;
Brunetti, M.; Renzoni, D.; Chakravarty, P.; Paolini, C.;
De Francesco, R.; Gallinari, P.; Steinkuhler, C.; Di
Marco, S., Proc. Natl. Acad. Sci. USA 2004, 101, 15064.
67.Schuetz, A.; Min, J.; Allali-Hassani, A.; Schapira, M.;
Shuen, M.; Loppnau, P.; Mazitschek, R.; Kwiatkowski,
N.P.; Lewis, T.A.; Maglathin, R.L.; McLean, T. H.;
Bochkarev, A.; Plotnikov, A.N.; Vedadi, M.; Arrowsmith,
C.H., J Biol Chem, 2008, 283, 11355.
68.You, L.; Feng, S.; An, R.; Wang, X., Nat. Prod. Commun.,
2009, 4, 297.
69.Huang, R.L.; Chen, C.C.; Huang, Y.L.; Hsieh, D.J.; Hu,
C.P.; Chen, C.F.; Chang, C., Hepatology, 1996, 24, 508.
70.Zhang, Q.; Qin, L.; He, W.; Van Puyvelde, L.; Maes, D.;
Adams, A.; Zheng, H.; De Kimpe, N., Planta Med., 2007,
73, 13.
71.Fujioka, T.; Furumi, K.; Fujii, H.; Okabe, H.; Mihashi,
K.; Nakano, Y.; Matsunaga, H.; Katano, M.; Mori, M.,
Chem. Pharm. Bull., 1999, 47, 96.
72.Kuo, P.L.; Hsu, Y.L.; Chang, C.H.; Chang, J.K., J.
Pharmacol. Exp. Ther., 2005. 314, 1290.
73.Tosun, A.; Akkol, E.K.; Yesilada, E., Z. Naturforsch.
C., 2009, 64, 56.

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系統識別號 U0007-1007201015250200
論文名稱(中文) 壹、N1-芳香基和苯甲基-4,5,6-三甲氧基吲哚為新穎微管蛋白聚合抑制劑之合成和結構與活性關係的研究 貳、開發苯磺醯取代之5-6騈環雜環為新穎組蛋白去乙醯酶抑制劑的研究
論文名稱(英文) I. Synthesis and Structure-Activity Relationships of N1-Aryl and Benzyl-4,5,6-Trimethoxyindoles as Novel Classes of Tubulin Polymerization Inhibitors II. Discovery of Benzenesulfonyl-Substituted Five-Six Fused Heterocycles as Novel Classes of Potent Histone Deacetylase Inhibitors
校院名稱 臺北醫學大學
系所名稱(中) 藥學研究所
系所名稱(英) Graduate Institute of Pharmacy
學年度 98
學期 2
出版年 99
研究生(中文) 賴玫蓉
學號 D301095004
學位類別 博士
語文別 中文
口試日期 2010-06-23
論文頁數 402頁
口試委員 委員-林仁混
委員-張俊彥
委員-李慶國
委員-林本元
指導教授-劉景平
關鍵字(中) 微管蛋白聚合抑制劑
組蛋白去乙醯酶
抑制劑
關鍵字(英) tubulin polymerization inhibitor
histone deacetylase inhibitor
學科別分類
中文摘要 第壹部分:N1-芳香基和苯甲基-4,5,6-三甲氧基吲哚為新穎微管蛋白聚合抑制劑之合成和結構與活性關係的研究

Combretastatin A-4結構的A、B環間之順式雙鍵對抗癌活性極為重要,若以醯胺鍵取代後則完全喪失活性,我們利用生物等效性的概念對I-217加上一個五元雜環,增加A環和醯胺基之間的固定性來限制其轉動,合成一系列1-芳醯基、苯甲基和苯磺醯基取代之4,5,6-三甲氧基吲哚和二氫吲哚化合物,對人類五種癌細胞 (口腔上皮細胞癌細胞KB、非小細胞肺癌細胞H460、結腸直腸癌細胞HT29、胃癌細胞MKN45和多重抗藥性癌細胞KB-vin10)進行實驗,結果發現:只有1-苯甲基取代之4,5,6-三甲氧基吲哚具有不錯的抗癌活性,於是進一步對B環進行化學修飾,其中I-225和I-226均呈現極強的抗癌活性 (平均IC50 = 26和27 nM),我們將抗癌活性佳的化合物進行抑制微管聚合試驗,I-225 ~ I-227的IC50分別為3.5、2.6、3.6 μM,皆比秋水仙素佳 (IC50 = 4.3 μM);於是我們保留4,5,6-三甲氧基吲哚,於N1位置直接導入不同取代基之苯環,其中I-256具有不錯的抗癌活性 (平均IC50 = 79 nM),為了改善其活性,將苯環置換成5-6騈環雜環或6-6騈環雜環,其中4位取代之吲哚 (I-239)和6位取代之奎寧 (I-244)均具強細胞毒性 (平均IC50 = 38和23 nM),我們選擇抗癌活性佳的化合物進行抑制微管聚合試驗,I-240和I-244的IC50均為2.7 μM,I-239的IC50為1.7 μM,與CA4相當 (IC50 = 1.7 μM)。以上實驗結果證明:本篇論文設計與合成出一系列N-苯甲基和芳香基取代之4,5,6-三甲氧基吲哚為新穎骨架的強微管聚合抑制劑。

第貳部分:開發苯磺醯基取代之5-6騈環雜環為新穎組蛋白去乙醯酶抑制劑的研究

由文獻得知,N-羥基丙烯醯胺常見於組蛋白去乙醯酶抑制劑的結構中,加上吲哚結構常出現於抗癌化合物中,本篇論文嘗試將此二結構組合起來,合成一系列苯磺醯基和N-羥基丙烯醯胺取代之吲哚化合物,對人類四種癌細胞 (包括肺癌細胞A549、乳癌細胞MDA-MB-231、肝癌細胞Hep-3B和HA22T)進行實驗,其中唯有II-88才具有抗癌活性,平均IC50為0.75 μM,將其苯磺醯基置換成苯甲醯基或苯甲基後均使抗癌活性喪失,若縮短或增加其鋅離子結合基與吲哚間的距離,也使抗癌活性喪失,若對其苯磺醯基進行化學修飾,導入不同的推拉電子基團合成II-94 ~ II-98,只有II-97喪失活性,其他化合物的活性 (平均IC50為0.65 ~ 1.35 μM)比SAHA (平均IC50為1.57 μM)強1.2 ~ 2.4倍;若將II-88的吲哚主結構置換為二氫吲哚,II-102的抗癌活性 (平均IC50 = 0.45 μM)可增強1.7倍,同樣於苯磺醯基導入不同的推拉電子基團後,只有II-106喪失其活性,II-103 ~ II-105及II-107的活性 (平均IC50為0.29 ~ 0.57 μM)比SAHA強2.8 ~ 5.4倍,由結構和活性的關係分析:二氫吲哚類化合物的抗癌活性比吲哚類化合物的活性強,於苯磺醯基導入4’-甲氧基或3’,4’-二甲氧基後可增強活性;將II-88和II-102對前列腺癌細胞PC3分別進行MTT assay和western blot,二化合物均對前列腺癌細胞PC3的存活率具有劑量相關及時間相關的現象,也證明均為組蛋白去乙醯酶抑制劑,對HDAC-1、HDAC-3和HDAC-6都有抑制能力。因此本篇論文設計與合成出一系列1-苯磺醯基取代之5-6騈環雜環為新穎之組蛋白去乙醯酶抑制劑。
英文摘要 Part I: Synthesis and Structure-Activity Relationships of N1-Aryl and Benzyl-4,5,6-Trimethoxyindoles as Novel Classes of Tubulin Polymerization Inhibitors

It has known that the olefin double bond of the cis-stilbene in combretastain A-4 is important for activity and the replacement of the double bond with an amide moiety resulted in dramatic loss of activity. Herein we utilized I-217 as a base to design the heterocycle derivatives of CA-4 to improve activity by a restricted methodology linking the amide group and A-ring via a five-member ring to introduce a series of 1-aroyl, 1-benzyl, and 1-benzenesulfonyl substituted 4,5,6-trimethoxyindoles and indolines. The antiproliferative activities against five human cancer cell lines showed that the 4,5,6-trimethoxyindole core with a N1-benzyl substitution imparts good cellular growth inhibitory activity. In an effort to improve activity, various substitutions was introduced to B-ring. I-225 and I-226 showed potent activity with mean IC50 values of 26 and 27 nM, respectively, and also exhibited substantial antitubulin activities with IC50 values of 2.6 and 3.5 μM, respectively, better than colchicine (IC50 = 4.3 μM). Further, we introduced various substituted phenyl group at N1 position of 4,5,6-trimethoxyindole and I-256 showed good activity with mean IC50 values of 79 nM. In an effort to further improve activity, we replaced the phenyl group with five-six or six-six fused heterocycles. I-239 and I-244 showed potent activity with mean IC50 values of 38 and 23 nM, respectively. They also exhibited antitubulin activities with IC50 values of 1.7 and 2.7 μM, respectively, comparable to CA-4 (IC50 = 1.7 μM). Therefore, we successfully designed and synthesized 1-aryl and benzyl-4,5,6-trimethoxyindoles as novel classes of potent antimitotic agents.

Part II: Discovery of Benzenesulfonyl-Substituted Five-Six Fused Heterocycles as Novel Classes of Potent Histone Deacetylase Inhibitors

From reviews, N-hydroxyacrylamide plays an important role for activity in HDAC inhibitors. In addition, indole ring usually exits in anticancer drugs. In this paper, we try to compose these two structures to introduce a serious of benzenesulfonyl and N-hydroxyacrylamide substituted indoles. The antiproliferative activities against four human cancer cell lines showed that II-88 imparts good activity with mean IC50 values of 0.75 μM. Whether the replacement of sulfonyl with carbonyl or methylene or the change of linker’s length would results in activity loss. After we introduced various subsitutions on benzenesulfonyl group to give II-94 ~ II-98, II-97 resulted in a dramatic decrease in activity and others showed potent activity with mean IC50 ranging from 0.65 ~ 1.35 μM, better than SAHA (mean IC50 = 1.57 μM) 1.2~2.4 –fold magnitude. Replacement of indole ring of II-88 with indoline to give II-102 resulted in an increased activity with mean IC50 values of 0.45 μM. We also introduced various substitutions to give II-103 ~ II-107. Only II-106 dramatically lost activity, others exhibited potent activity with mean IC50 ranging from 0.29 ~ 0.57 μM, better than SAHA 2.8 ~ 5.4-fold magnitude. The SAR studies showed that indolines has stronger potency than indoles and 3’,4’-dimethoxy and 4’-methoxy substitutions on the benzenesulfonyl group increase antiproliferative activity. From MTT assay and western blot results, both II-88 and II-102 showed time- and dose-dependent effects and potent inhibition on HDAC-1, 3, and 6 in PC3 cells. Therefore, we successfully designed and synthesized benzenesulfonyl five-six fused heterocycles as novel classes of potent histone deacetylase inhibitors.
論文目次 目錄
中文摘要.........................................................................................................................I
英文摘要......................................................................................................................III
謝誌...............................................................................................................................V
目錄............................................................................................................................VI
表目錄.......................................................................................................................XV
圖目錄..................................................................................................................... XVI
流程目錄................................................................................................................XXIII
縮寫對照表.............................................................................................................XXV

第壹部分:N1-芳香基和苯甲基-4,5,6-三甲氧基吲哚為新穎微管蛋白聚合抑制劑之合成和結構與活性關係的研究

壹、 緒論…………………….………………………………......................................1
1.1前言…………………………………………………….................................1
1.2抗癌藥物的分類…………………………..…………………………...........3
1.2.1 抗代謝物……………………………………………………………...3
1.2.2 烷化劑……………………………….………………………………..4
1.2.3 抗生素………………………………………………………………...6
1.2.4 拓撲異構酶抑制劑…………………………………………………...7
1.2.5 微管抑制劑…………………………………………………………...8
1.2.6 荷爾蒙拮抗劑………………………………………………………...8
1.2.7 其它抗癌藥物………………………………………………………...9
1.3微管的背景介紹...........................................................................................10
1.3.1 細胞週期.............................................................................................10
1.3.2 有絲分裂.............................................................................................11
1.3.3 微管的結構與功能.............................................................................12
1.4以微管為標靶的抗癌藥物...........................................................................13
1.4.1 微管去穩定劑.....................................................................................14
1.4.2 微管穩定劑.........................................................................................14
1.5 Combretastatin A-4 (CA-4)類化合物之研究概況.......................................15
1.5.1 Combretastatins之研究.......................................................................15
1.5.2血管標靶治療......................................................................................17
1.5.2.1小分子血管破壞劑..................................................................17
1.5.2.2直接與配體作用的血管破壞劑..............................................21
1.5.3 Combretastatin A-4衍生物..................................................................22
1.6 CombretastatinA-4類化合物之合成與活性................................................24
1.6.1 A-B環以單原子連結之類化合物.......................................................25
1.6.2 A-B環以雙原子連結之類化合物.......................................................31
1.6.2.1非環狀連結之類化合物..........................................................32
1.6.2.2非芳香環連結之類化合物......................................................36
1.6.2.3雜芳香環連結之類化合物......................................................39
1.6.2.4 5-6騈環雜環系統連結之類化合物.......................................43
1.6.3 A-B環以三原子連結之類化合物.......................................................44
1.6.3.1 1,3-位置雜環為連結之類化合物...........................................45
1.6.3.2 α, β-不飽合酮類為連結之類化合物......................................46
1.6.4 A-B環以四原子連結之類化合物.......................................................49
1.7修飾A環之combretastatin類化合物...........................................................50
1.8修飾B環之combretastatin類化合物...........................................................54
1.8.1多種取代基之苯環…………………………......................................54
1.8.2雜環......................................................................................................57
1.8.3芳香環..................................................................................................59
1.9吲哚化合物與Combretastatin A-4之結構和活性關係(SAR)之研究........64
1.9.1 2-芳醯基吲哚之衍生物......................................................................64
1.9.2 1-和3-芳醯基吲哚之衍生物...............................................................67
1.9.3 4-和5-芳醯基吲哚之衍生物...............................................................74

貳、 研究構想.............................................................................................................76
2.1以吲哚為主之化合物設計與抗癌活性的研究...........................................77
2.2本論文之藥物設計理念...............................................................................78
2.3合成方法之文獻回顧...................................................................................83
2.3.1 4,5,6-三甲氧基吲哚之文獻回顧........................................................83
2.3.2 4,5,6-Trimethoxy-1H-indole-2-carboxylic acid methyl ester之文獻
回顧.....................................................................................................84

參、 結果與討論.........................................................................................................85
3.1 4,5,6-三甲氧基吲哚類之化合物的合成研究.............................................85
3.1.1 1-芳醯基、苯甲基、苯磺醯基取代之4,5,6-三甲氧基吲哚化合物的
合成研究.............................................................................................85
3.1.2 1-芳醯基、苯甲基、苯磺醯基取代之4,5,6-三甲氧基二氫吲哚化
合物的合成研究.................................................................................89
3.1.3 N-phenylbenzamide和benzylphenylamine化合物的合成
研究...................................................................................................90
3.1.4 1-苯基取代之4,5,6-三甲氧基吲哚化合物的合成研究.....................91
3.1.5 1-吲哚基、苯呋喃基、奎寧基、喹唑啉基取代之4,5,6-三甲氧基
吲哚化合物的合成研究.....................................................................92
3.2 4,5,6-三甲氧基吲哚類化合物的抗癌活性之研究.....................................96
3.2.1 1-芳醯基、苯甲基、苯磺醯基取代之4,5,6-三甲氧基吲哚
(二氫吲哚) 化合物的抗癌活性.........................................................96
3.2.2 1-苯基、吲哚基、苯呋喃基、奎寧基、喹唑啉基取代之4,5,6-
三甲氧基吲哚化合物的抗癌活性.....................................................99
3.3微管蛋白競爭性試驗.................................................................................103
3.3.1 1-苯甲基取代之4,5,6-三甲氧基吲哚之微管蛋白競爭性試驗.......103
3.3.2 1-苯基、吲哚基、奎寧基取代之4,5,6-三甲氧基吲哚之微管蛋白
競爭性試驗.......................................................................................105
3.4結論.............................................................................................................106

第貳部分:開發苯磺醯取代之5-6騈環雜環為新穎組蛋白去乙醯酶抑制劑的研究

肆、 緒論...................................................................................................................109
4.1前言.............................................................................................................109
4.2組蛋白去乙醯酶的生物活性.....................................................................110
4.2.1組蛋白去乙醯酶的分類....................................................................111
4.2.2組蛋白去乙醯酶的位置....................................................................113
4.2.3組蛋白去乙醯酶的功能....................................................................114
4.3組蛋白去乙醯酶的結構研究.....................................................................118
4.4組蛋白去乙醯酶的藥理作用.....................................................................122
4.4.1組蛋白乙醯轉化酶和組蛋白去乙醯酶............................................122
4.4.2組蛋白去乙醯酶在癌症上的作用....................................................123
4.4.3組蛋白去乙醯酶在其他治療方向的作用........................................125
4.5組蛋白去乙醯酶抑制劑.............................................................................126
4.5.1組蛋白去乙醯酶抑制劑之作用機轉................................................128
4.5.2發展組蛋白去乙醯酶抑制劑成為抗癌藥物....................................128
4.5.3組蛋白去乙醯酶抑制劑的分類及其抗癌活性................................129
4.5.3.1短鏈脂肪酸............................................................................129
4.5.3.2 Hydroxamic acid之Trichostatin A……………………….130
4.5.3.3 Hydroxamic acid之SAHA……………………………..…..131
4.5.3.4 親電性酮類..........................................................................133
4.5.3.5 Aminobenzamide...................................................................134
4.5.3.6 環狀胜肽..............................................................................135
4.5.4組蛋白去乙醯酶抑制劑的非癌症治療潛力....................................137
4.6組蛋白去乙醯酶抑制劑之結構與活性的關係.........................................138
4.6.1 具長鏈連結之hydroxamic acid 類抑制劑.....................................139
4.6.2 具肉桂醯連結之hydroxamic acid 類抑制劑.................................145
4.6.3 具芳香環或雜芳香環連結之hydroxamic Acid 類抑制劑.............150
4.6.4 Thiols and Thiol Derivatives..............................................................157
4.6.5 2-Aminophenylamides.......................................................................161
4.6.6 酮類衍生物.......................................................................................168
4.6.7 其他...................................................................................................171
4.7組蛋白去乙醯酶抑制劑的選擇性.............................................................173
4.8具選擇性的class I和class II組蛋白去乙醯酶抑制劑之體內實驗活
性.……………………………………………………………………..174
4.9組蛋白去乙醯酶抑制劑的臨床實驗.........................................................176

伍、 研究構想...........................................................................................................180
5.1已進入臨床試驗之hydroxamic acid類組蛋白去乙醯酶抑制劑的結
構分析.........................................................................................................180
5.2本論文之藥物設計理念.............................................................................181

陸、 結果與討論.......................................................................................................187
6.1 Hydroxamaic acid類化合物的合成研究...................................................187
6.1.1 1-Benzenesulfonyl-3,4,5,6,7-(N-hydroxyacrylamide)indoles化合物
的合成研究....................................................................................187
6.1.2 1-Aroyl, 1-benzyl, 1-phenyl-5-(N-hydroxyacrylamide)-indoles化
合物的合成研究.............................................................................190
6.1.3 3-Benzenesulfonyl-5, 6 and 7-(N-hydroxyacrylamide)-indoles化
合物的合成研究.............................................................................191
6.1.4 1-Benzenesulfonyl-5-(N-hydroxyacrylamide)-indolines化合物的
合成研究.........................................................................................193
6.1.5 1-Benzenesulfonyl-5-hydroxamic acid-indole and indoline化合
物的合成研究.................................................................................194
6.1.6 1-Benzenesulfonyl-5-(amide-but-2-enoic acid hydroxyamide)-
indole化合物的合成研究................................................................196
6.1.7 1-Benzenesulfonyl-5-(2-aminophenylamide)-indole and indoline
化合物的合成研究.........................................................................197
6.2 Hydroxamaic acid類化合物的藥理活性研究...........................................199
6.2.1 Hydroxamaic acid類化合物的抗癌活性..........................................199
6.2.2化合物II-88和II-102對組蛋白去乙醯酶相關的蛋白質之抑制
能力...............................................................................................203
6.3結論.............................................................................................................204

柒、 實驗部分...........................................................................................................206
7.1一般實驗方法.............................................................................................206
7.2第壹部分化合物之實驗方法與光譜資料.................................................209
7.2.1 化合物I-217之合成.......................................................................209
7.2.2 化合物I-218之合成.......................................................................210
7.2.3 化合物I-219之合成.......................................................................211
7.2.4 化合物I-220之合成.......................................................................211
7.2.5 化合物I-221之合成.......................................................................212
7.2.6 化合物I-222之合成.......................................................................213
7.2.7 化合物I-223之合成.......................................................................214
7.2.8 化合物I-224之合成.......................................................................215
7.2.9 化合物I-225之合成.......................................................................216
7.2.10 化合物I-226之合成......................................................................217
7.2.11 化合物I-227之合成......................................................................218
7.2.12 化合物I-228之合成......................................................................219
7.2.13 化合物I-229之合成......................................................................219
7.2.14 化合物I-230之合成......................................................................220
7.2.15 化合物I-231之合成......................................................................221
7.2.16 化合物I-232之合成......................................................................222
7.2.17 化合物I-233之合成......................................................................222
7.2.18 化合物I-234之合成......................................................................224
7.2.19 化合物I-235之合成......................................................................225
7.2.20 化合物I-236之合成......................................................................227
7.2.21 化合物I-237之合成......................................................................229
7.2.22 化合物I-238之合成......................................................................229
7.2.23 化合物I-239之合成......................................................................230
7.2.24 化合物I-240之合成......................................................................231
7.2.25 化合物I-241之合成......................................................................233
7.2.26 化合物I-242之合成......................................................................235
7.2.27 化合物I-243之合成......................................................................235
7.2.28 化合物I-244之合成......................................................................236
7.2.29 化合物I-245之合成......................................................................237
7.2.30 化合物I-246之合成......................................................................238
7.2.31 化合物I-247之合成......................................................................239
7.2.32 化合物I-248之合成......................................................................242
7.2.33 化合物I-251之合成......................................................................243
7.2.34 化合物I-254之合成......................................................................244
7.2.35 化合物I-257之合成......................................................................246
7.2.36 化合物I-258之合成......................................................................246
7.3第貳部分化合物之實驗方法與光譜資料.................................................248
7.3.1 化合物II-86之合成.......................................................................248
7.3.2 化合物II-87之合成.......................................................................250
7.3.3 化合物II-88之合成.......................................................................253
7.3.4 化合物II-89之合成.......................................................................255
7.3.5 化合物II-90之合成.......................................................................258
7.3.6 化合物II-91之合成.......................................................................261
7.3.7 化合物II-92之合成.......................................................................263
7.3.8 化合物II-93之合成.......................................................................266
7.3.9 化合物II-94之合成.......................................................................269
7.3.10 化合物II-95之合成......................................................................272
7.3.11 化合物II-96之合成......................................................................274
7.3.12 化合物II-97之合成.....................................................................277
7.3.13 化合物II-98之合成......................................................................278
7.3.14 化合物II-99之合成......................................................................280
7.3.15 化合物II-100之合成....................................................................284
7.3.16 化合物II-101之合成....................................................................287
7.3.17 化合物II-102之合成....................................................................290
7.3.18 化合物II-103之合成....................................................................295
7.3.19 化合物II-104之合成....................................................................298
7.3.20 化合物II-105之合成....................................................................302
7.3.21 化合物II-106之合成....................................................................305
7.3.22 化合物II-107之合成....................................................................306
7.3.23 化合物II-108之合成....................................................................310
7.3.24 化合物II-109之合成....................................................................312
7.3.25 化合物II-110之合成....................................................................314
7.3.26 化合物II-111之合成....................................................................316
7.3.27 化合物II-112之合成....................................................................317

捌、 參考文獻...........................................................................................................318

附錄一、化合物之氫核磁共振光譜..........................................................................341
附錄二、發表論文......................................................................................................402
參考文獻 捌、參考文獻

1. http://www.who.int/mediacentre/factsheets/fs297/en/index.html
2. http://www.doh.gov.tw (行政院衛生署最新發布統計資料)
3. Williams, D. A.; Lemke, T. L. Foye’s Principles of Medicinal Chemistry; Lippincott Williams & Wilkins; 5th edition; 2002; 924-929.
4. Lipp, H. P. Anticancer Drug Toxicity: Prevention, Management, and Clinical Pharmacokinetics; Marcel Dekker Inc.; New York; 1999; 11-201.
5. Lee K. H. Current developments in the discovery and design of new drug candidates from plant natural product leads. J. Nat. Prod. 2003; 66, 1022-1037.
6. Heald, R.; Nogales, E. Microtubule dynamics. J. Cell Sci. 2002, 115, 3-4.
7. Mahindroo, N.; Liou, J. P.; Chang, J. Y.; Hsieh, H. P. Antitubulin agents for the treatment of cancer a medicinal chemistry update. Expert Opin. Ther. Patents 2006, 16, 647-691.
8. Dancey, J.; Sausville, E. A. Issues and progress with protein kinase inhibitors for cancer treatment. Nat. Rev. Drug Discov. 2003, 2, 296-313.
9. Arora, A.; Scholar, E. M. Role of tyrosine kinase inhibitors in cancer therapy. J. Pharmaco. Exp. Therapeut. 2005, 315, 971-979.
10. http://www.physiomics-plc.com/cell_cycle.htm.
11. Jordan, A.; Hadfield, J. A.; Lawrence, N. J.; McGown, A. T. Tubulin as a target for anticancer drugs: Agents which interact with the mitotic spindle. Med. Res. Rev. 1998, 18, 259-296.
12. http://www.le.ac.uk/ge/genie/vgec/he/cellcycle.html.
13. Jordan, M. A.; Wilson, L. Microtubules as a target for anticancer drugs. Nat. Rev. Cancer 2004, 4, 253-265.
14. Lodish, H; Scott, M. P.; Matsudaira, P.; Darnell, J.; Zipursky, L.; Kaiser, C. A.; Berk, A.; Krieger, M.; Molecular Cell Biology; W. H. Freeman; 5th edition; 2003.
15. Dumont, R.; Broeei, A.; Chignell, C. F.; Quinn, F. R.; Suffness, M. A. Novel synthesis of colchicines and analogues from thiocolchine and congeners:
reevaluation of colchicines as a potential antitumor agent. J. Med. Chem. 1987, 30, 732-735.
16. Jordan, M. A.; Thrower, D. & Wilson, L. Mechanism of inhibition of cell proliferation by Vinca alkaloids. Cancer Res. 1991, 51, 2212-2222.
17. Mujagic, H.; Conger, B. M.; Smith, C. A.; Occhipinti, S. J.; Schuette, W. H.; Shackney, S. E. Schedule dependence of vincristine lethality in sarcoma 180 cells following partial synchronization with hydroxyurea. Cancer Res. 1983, 43, 3598-3603.
18. Manfriedi, J. J.; Horwitz, S. B. Taxol: an antimitotic agent with a new mechanism of action. Pharmacol. Ther. 1984, 25, 83.
19. Pettit, G. R.; Cragg, G. M.; Herald, D. L.; Schmidt, J. M. Isolation and structure of Combretastatin. Can. J. Chem. 1982, 60, 1374-1376.
20. Pettit, G. R.; Singh, S. B.; Boyd, M. R.; Hamel, E.; Pettit, R. K.; Schmidt, J. M.; Hogan, F. Isolation and synthesis of combretastatins A-4, A-5, and A-6. J. Med. Chem. 1995, 38, 1666-1672.
21. Nam, N. H. Combretastatin A-4 analogues as antimitotic antitumor agents. Curr. Med. Chem. 2003, 10, 1697-1722.
22. Cirla, A; Mann, J. Combretastatins: from natural products to drug discovery. Nat. Prod. Rep. 2003, 20, 558-564.
23. Prinz, H. Recent advances in the field of tubulin polymerization inhibitors. Expert Rev. Anticancer Ther. 2002, 2, 695-708.

24. Denekamp, J. Endothelial cell attack as a novel approach to cancer therapy. Cancer Topics 1986, 6, 6-8.
25. Denekamp, J. Vascular attack as a therapeutic strategy for cancer. Cancer Metastasis Rev. 1990, 9, 267-282.
26. Thorpe, P. E. Vascular targeting agents as cancer therapeutics. Clin. Cancer Res. 2004, 10, 415-427.
27. Tozer,G. M.; Kanthou, C.; Baguley, B. C. Disrupting tumour blood vessels. Nat. Rev. Cancer 2005, 5, 423-435.
28. Tozer, G. M.; Kanthou, C.; Parkins C. S.; Hill, S. A. The biology of the combretastatins as tumour vascular targeting agents. Int. J. Exp. Pathol. 2002, 83, 21-38.
29. Lippert III, J. W. Vascular disrupting agents. Bioorg. Med. Chem. 2007, 15, 605-615.
30. Chaplin, D. J.; Hill, S. A. The development of Combretastatin A4 phosphate as a vascular targeting agent. Int. J. Radia. Oncol. Biol. Phys. 2002, 54, 1491-1496.
31. http://clinicaltrials.gov/
32. http://www.oxigene.com/
33. Pettit, G. R.; Lippert, J. W. Syntheses of the combretastatin A-1 and combretastatin B-1 prodrugs. Anticancer Drug Des. 2000, 15, 203-216.
34. Pilat M. J.; LoRusso, P. M. Vascular disrupting agents. J. Cell. Biochem. 2006, 99, 1021-1039.
35. Hsieh, H. P.; Liou, J. P.; Mahindroo, N. Pharmaceutical sesign of antimitotic agents based on combretastatins. Curr. Pharm. Des. 2005, 11, 1655-1677.
36. Tron, G. C.; Pirali, T.; Sorba, G.; Pagliai, F.; Busacca, S.; Genazzani, A. A. Medicinal chemistry of combretastatin A4: present and future directions. J. Med. Chem., 2006, 49, 3033–3044.
37. Kaffy, J.; Pontikis, R.; Florent, J. C.; Monnert, C. Synthesis and biological evaluation of vinylogous combretastatin A-4 derivatives. Org. Biomol. Chem. 2005, 3, 2657-2660.
38. Cushman, M.; Nagarathnam, D.; Gopal, D.; Chakraborti, A. K.; Lin, C. M.; Hamel, E. Synthesis and evaluation of stilbene and dihydrostibene derivatives as potential anticancer agents that inhibit tubulin polymerization. J. Med. Chem. 1991, 34, 2579-2588.
39. Cushman, M.; Nagarathnam, D.; Gopal, D.; He, H. M.; Lin, C. M.; Hamel, E. Synthesis and evaluation of (Z)-1-(4-Methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)ethane as potential cytotoxic and antimitotic agents. J. Med. Chem. 1992, 35, 2239-2306.
40. Pettit, G. R.; Toki, B. E.; Herald, D. L.; Verdier-Pinard, P.; Boyd, M. R.; Hamel, E.; Pettit, R. K. Antineoplastic agents. 379. Synthesis of phenstatin phosphate. J. Med. Chem. 1998, 41, 1688-1689.
41. Pettit, G. R.; Lippert III, J. W.; Herald, D. L. A pinacol rearrangement/oxidation synthetic route to hydrophenstatin. J. Org. Chem. 2000, 65, 7438-7444.
42. Pettit, G. R.; Grealish, M. P.; Herald, D. L.; Boyd, M. R.; Hamel, E.; Pettit, R. K. Antineoplastic agents. 443. Synthesis of the cancer cell growth inhibitor hydroxyphenstatin and its sodium diphosphate prodrug. J. Med. Chem. 2000, 43, 2731-2737.
43. Liou, J. P.; Chang, C. W.; Song, J. S.; Yang, Y. N.; Yeh, C. F.; Tseng, H. Y.; Lo, Y. K.; Chang, Y. L.; Chang, C. M.; Hsieh, H. P. Synthesis and structure-activity relationship of 2-aminobenzophenone derivatives as antimitotic agents. J. Med. Chem. 2002, 45, 2556-2562.
44. Liou, J. P.; Chang, J. Y.; Chang, C. W.; Chang, C. Y.; Mahindroo, N.; Kuo, F. M.; Hsieh, H. P. Synthesis and structure-activity relationship of 3-aminobenzophenone as antimitotic agents. J. Med. Chem. 2004, 47, 2897-2905.
45. Hsieh, H. P.; Liou, J. P.; Lin, Y. T.; Mahindroo, N.; Chang, J. Y.; Yang, Y. N.; Chern, S. S.; Tan, U. K.; Chang, C. W.; Chen, T. W.; Lin, C. H.; Chang, Y. Y.; Wang, C. C. Structure-activity and crystallographic analysis of benzophenone derivatives-the potential anticancer agents. Bioorg. Med. Chem. Lett. 2003, 13, 101-105.
46. Lawrence, N. J.; Rennison, D.; Woo, M.; McDown, A. T.; Hadfield, J. A. Antimitotic and cell growth inhibitory properties of combretastatin A-4-like ethers. Bioorg. Med. Chem. Lett. 2001, 11, 51-54.
47. Getahun, Z.; Jurd, L.; Chu, P. S.; Lin, C. M.; Hamel, E. Synthesis of alkoxy-substituted diaryl compounds and correlation of ring separation with inhibition of tubulin polymerization: differential enhancement of inhibitory effects under suboptimal polymerization reaction conditions. J. Med. Chem. 1992, 35, 1058-1067.
48. Pettit, G. R.; Toki, B. E.; Herald, D. L.; Boyd, M. R.; Hamel, E.; Pettit, R. K.; Chapuis, J. C. Antineoplastic agents. 410. Asymmetric hydroxylation of trans-combretastatin A-4. J. Med. Chem. 1999, 42, 1459-1465.
49. Iwasaki, S.; Shirai, R. Natural organic compounds that affect to microtubule functions: syntheses and structure-activity relationships of combretastatins, curacin A and their analogs as the colchicines-site ligands on tubulin. Yakugaku Zasshi 2000, 120, 875-889.
50. Medarde, M.; Ramos, S.; Caballero, E.; Lamamie de Clairac, R. P.; Lopez, J. L.; Gravalos, D. G.; Feliciano, A. Synthesis and antineoplastic activity of combretastatin analogues: Heterocombretastatins. Eur. J. Med. Chem. 1998, 33, 71-77.

51. Li, Q.; Sham, H. L. Discovery and development of antimitotic antitumor agents that inhibit tubulin polymerization for the treatment of cancer. Expert Opin. Ther. Pat. 2002, 12, 1663-1702.
52. Shirai, R.; Toukuda, K.; Koiso, Y.; Iwasaki, S. Synthesis and anti-tubulin activity of aza-combretastatins. Bioorg. Med. Chem. Lett. 1994, 4, 699-704.
53. Gwaltney II, S. L.; Imade, H. M.; Barr, K. J.; Li, Q.; Gehrke, L.; Credo, R. B.; Warner, R. B.; Lee, J. Y.; Kovear, P.; Wang, J.; Nukkala, M. A.; Zielinski, N. A.; Frost, D.; Ng, S. C. Novel sulfonate analogues of combretastatin A-4: potent antimitotic agents. Bioorg. Med. Chem. Lett. 2001, 11, 871-874.
54. Ohsumi, K.; Nakagawa, R.; Fukuda, Y.; Hatanaka, T.; Morinaga, Y.; Nihei, Y.; Ohishi, K.; Suga, Y.; Akiyama, Y.; Tsuji, T. Novel combretastatin analogues effective against murine solid tumors: design and structure-activity relationships. J. Med. Chem. 1998, 41, 3022-3032.
55. Hadfield, J. A.; Gaukroger, K.; Hirst, N.; Wetston, A. P.; Lawrence, N. J.; McGown, A. T. Synthesis and evaluation of double bond substituted combretastatins. Eur. J. Med. Chem. 2005, 40, 529-541.
56. Alloatti, D.; Giannini, G.; Cabri, W.; Lustrati, I.; Marzi, M.; Ciacci, A.; Gallo, G.; Tinti, M. O.; Marcellni, M.; Riccioni, T.; Guglielmi, M. B.; Carminati, P.; Pisano, C. Synthesis and biological activity of fluorinate combretastatin analogues. J. Med. Chem. 2008, 51, 2708-2721.
57. Shirai, R.; Takayama, H.; Nishikaea, A.; Koiso, Y.; Hashimoto, Y. Asymmetric synthesis of antimitotic combretadioxolane with potent antitumor activity against multi-drug resistant cells. Bioorg. Med. Chem. Lett. 1998, 8, 1997-2000.
58. Shirai, R.; Okabe, T.; Iwasaki, S. Synthesis of conformationally restricted combretastatins. Heterocycles 1997, 46, 145-148.

59. Nam, N. H.; Kim, Y.; You, Y. J.; Hong, D. H.; Kim, H. M.; Ahn, B. Z. Synthesis and anti-tumor activity of novel combretastatin: combretocyclopentenones and related analogues. Bioorg. Med. Chem. Lett. 2002, 12, 1955-1958.
60. Flynn, B. L.; Flynn, G. P.; Hamel, E.; Jung, M. K. The synthesis and tubulin binding activity of thiophene-based analogues of combretastatin A-4. Bioorg. Med. Chem. Lett. 2001, 11, 2341-2343.
61. Kim, Y.; Nam, N. H.; You, Y. J.; Ahn, B. Z. Synthesis and cytotoxicity of 3, 4-diaryl-2(5H)-furanones. Bioorg. Med. Chem. Lett. 2002, 12, 719-722.
62. Nam, N. H.; Kim, Y.; You, Y. J.; Hong, D. H.; Kim, H. M.; Ahn, B. Z. Water soluble prodrugs of the antitumor agent 3-[(3-amino-4- methoxy)phenyl]-2-(3, 4, 5-trimethoxyphenyl)cyclopent-2-ene-1- one. Bioorg. Med. Chem. 2003, 11, 1021-1029.
63. Nam, N. H.; Kim, Y.; You, Y. J.; Hong, D. H.; Kim, H. M.; Ahn, B. Z. Combretoxazolones: synthesis, cytotoxicity and antitumor activity. Bioorg. Med. Chem. Lett. 2001, 11, 3073-3076.
64. Simoni, D.; Grisolia, G.; Giannini, G.; Roberti, M.; Rondanin, R.; Piccagli, L.; Baruchello, R.; Rossi, M.; Romagnoli, R.; Invidiata, F. P.; Grimaudo, S.; Jung, M. K.; Hamel, E.; Gebbia, N.; Crosta, L.; Abbadessa, V.; Cristina, A. D.; Dusonchet, L.; Meli, M.; Tolomeo, M. Heterocyclic and phenyl double-bond-locked combretastatin analogues possessing potent apoptosis-inducing activity in HL60 and in MDR cell lines. J. Med. Chem. 2005, 48, 723-736.
65. Gurjar, M. K.; Wakharkar, R. D.; Singh, A. T.; Jaggi, M.; Borate, H. B.; Shinde, P. D.; Verma, R.; Rajendran, P.; Dutt, S.; Singh, G.; Sanna, V. K.; Singh, M. K.; Srivastava, S. K.; Mahajan, V. A.; Jadhav, V. H.; Dutta, K.; Krishnan, K.; Chaudhary, A.; Agarwal, S. K.; Mukherjee, R.; Burman, A. C. Synthesis and evaluation of 4/5-hydroxy-2,3-diaryl(substituted)-cyclopent-2-en-1-ones as cis-restricted analogues of combretastatin A-4 as novel anticancer agents. J. Med. Chem. 2007, 50, 1744-1753.
66. Ohsumi, K.; Hatanaka, T.; Fujita, K.; Nakagawa, R.; Fukuda, Y.; Nihei, Y.; Suga, Y.; Morinaga, Y.; Akiyama, Y.; Tsuji, T. Syntheses and antitumor activity of cis-restricted combretastatins: 5-membered heterocyclic analogues. Bioorg. Med. Chem. Lett. 1998, 8, 3153-3158.
67. Wang, L.; Woods, K.W.; Li, Q.; Barr, K.J.; McCroskey, R.W.; Hannick, S. M.; Gherke, L.; Credo, R. B.; Hui, Y. H.; Marsh, K.; Warner, R.; Lee, J. Y.; Mozng, N. Z.; Frost, D.; Rosenberg, S. H.; Sham, H. L. Potent, orally active heterocycle-based combretastatin A-4 analogues: synthesis, structure-activity relationship, pharmacokinetics, and in vivo antitumor activity evaluation. J. Med. Chem. 2002, 45, 1697-1711.
68. Zhang, Q.; Peng, Y.; Wang, X. I.; Keenan, S. M.; Arora, S.; Welsh, W. J. Highly potent triazole-based tubulin polymerization inhibitors. J. Med. Chem. 2007, 50, 749-754.
69. Medarde, M.; Ramos, A. C.; Caballero, E.; Pelaez-Lamamie de Clairac, R.; Lopez, J. L.; Gravalos, D. G.; Feliciano. A. S. Synthesis and pharmacological activity of diarylindole derivatives. Cytotoxic agents based on combretastatins. Bioorg. Med. Chem. Lett. 1999, 9, 2303-2308.
70. Flynn, B. L.; Hamel, E.; Jung, M. K. One-pot synthesis of benzo[b]furan and indole inhibitors of tubulin polymerization. J. Med. Chem. 2002, 45, 2670-2673.
71. Pinney, K. G.; Bounds, A. D.; Dingeman, K. M.; Mocharla, V. P.; Pettit, G. R.; Bai, R.; Hamel, E. A new anti-tubulin agent containing the benzo[b]thiophene ring system. Bioorg. Med. Chem. Lett. 1999, 9, 1081-1086.
72. Wu-Wong, J. R.; Alder, J. D.; Alder, L.; Burns, D. J.; Han, E. K.; Credo, B.; Tahir, S. K.; Dayton, B. D.; Ewing, P. J.; Chiou. W. J. Identification and characterization of A-105972, an antineoplastic agent. Cancer Res. 2001, 61, 1486-1492.
73. Tahir, S. K.; Han, E. K.; Credo, B.; Jae, H. S.; Pietenpol, J. A.; Scatena, C. D.; Wu-Wong, J. R.; Frost, D.; Sham, H.; Rosenberg, S. H.; Ng, S. C. A-204197, a new tubulin-binding agent with antimitotic activity in tumor cell lines resistant to known microtubule inhibitors. Cancer Res. 2001, 61, 5480-5485.
74. Szczepankiewicz, B. G.; Liu, G.; Jae, H. S.; Tasker, A. S.; Gunawardana, I. W.; von Geldern, T. W.; Gwaltney II, S. L.; Wu-Wong, J. R.; Gehrke, L.; Chiou, W. J.; Credo, R. B.; Alder, J. D.; Nukkala, M. A.; Zielinski, N. A.; Jarvis, K.; Mollison, K. W.; Frost, D. J.; Bauch, J. L.; Hui, Y. H.; Claiborne, A. K.; Li, Q.; Rosenberg, S. H. New antimitotic agents with activity in multi-drug-resistant cell lines and in vivo efficacy in murine tumor models. J. Med. Chem. 2001, 44, 4416-4430.
75. Li, Q.; Woods, K. W.; Claiborne, A.; Gwaltney, S. L. 2nd; Barr, K. J.; Liu, G.; Gehrke, L.; Credo, R. B.; Hui, Y. H.; Lee, J.; Warner, R. B.; Kovar, P.; Nukkala, M. A.; Zielinski, N. A.; Tahir, S. K.; Fitzgerald, M.; Kim, K. H.; Marsh, K.; Frost, D.; Ng, S. C.; Rosenberg, S.; Sham, H. L. Synthesis and biological evaluation of 2-indolyloxazolines as a new class of tubulin polymerization inhibitors. Discovery of A-289099 as an orally active antitumor agent. Bioorg. Med. Chem. Lett. 2002, 12, 465-469.
76. Flynn, B. L.; Verdier-Pinard, P.; Hamel, E. A novel palladium-mediated coupling approach to 2, 3-disubstituted benzo(b)thiophenes and its application to the synthesis of tubulin binding agents. Org. Lett. 2001, 3, 651-654.
77. Chen, Z.; Mocharla, V. P.; Farmer, J. M.; Pettit, G. R.; Hamel, E.; Pinney, K. G. Preparation of new anti-tubulin ligands through a dual-mode, addition-elimination reaction to a bromo-substituted alpha, betaunsaturated sulfoxide. J. Org. Chem. 2000, 65, 8811-8815.
78. Ducki, S.; Forrest, R.; Hadfield, J. A.; Kendall, A.; Lawrence, N. J.; McGown, A. T.; Rennison, D. Potent antimitotic and cell growth inhibitory properties of substituted chalcones. Bioorg. Med. Chem. Lett. 1998, 8, 1051-1056.
79. Beutler, J. A.; Hamel, E.; Vlietinck, A. J.; Haemers, A.; Rajan, P.; Roitman, J. N.; Cardellina II, J. H.; Boyd, M. R. Structure-activity requirements for flavone cytotoxicity and binding to tubulin. J. Med. Chem. 1998, 41, 2333-2338.
80. Lichius, J. J.; Thoison, O.; Montagnac, A.; Pais, M.; Gueritte-Voegelein, F.; Sévenet, T.; Cosson, J. P.; Hadi, A. H. A. Antimitotic and cytotoxic flavonols from Zieridium pseudobtusifolium and Acronychia porteri. J. Nat. Prod. 1994, 57, 1012-1016.
81. Shi, Q.; Chen, K.; Li, L.; Chang, J. J.; Autry, C.; Kozuka, M.; Konoshima, T.; Estes, J. R.; Lin, C. M.; Hamel, E.; McPhail, A. T.; McPhail, D. R.; Lee, K. H. Antitumor agents, 154. Cytotoxic and antimitotic flavonols from Polanisia dodecandra. J. Nat. Prod. 1995, 58, 475-482.
82. Gaukroger, K.; Hadfield, J. A.; Lawrence, N. J.; Nolan, S.; McGown, A. T. Structural requirements for the interaction of combretastatins with tubulin: how important is the trimethoxy unit? Org. Biomol. Chem. 2003, 1, 3033-3037.
83. Pettit, G. R.; Minardi, M. D.; Rosenberg, H. J.; Hamel, E.; Bibby, M. C.; Martin, S. W.; Jung, M. K.; Pettit, R. K.; Cuthbertson, T. J.; Chapuis, J. C. Antineoplastic Agent. 509. Synthesis of fluorcombstatin phosphate and related 3-halostilbenes. J. Nat. Prod. 2005, 68, 1450-1458.
84. Pettit, G. R.; Anderson, C. R.; Herald, D. L.; Jung, M. K.; Lee, D. J.; Hamel, E.; Pettit, R. K. Antineoplastic agents. 487. synthesis and biological evaluation of the antineoplastic agent 3,4-methylenedioxy-5,4’-dimethoxy-3’-amino-Z-stilbene and derived amino acid amides. J. Med. Chem. 2003, 46, 525-531.

85. Simoni, D.; Romagnoli, R.; Baruchello, R.; Rondanin, R.; Grisolia, G.; Eleopra, M.; Rizzi, M.; Tolomeo, M.; Giannini, G.; Alloatti, D.; Castorina, M.; Marcellini, M.; Pisano, C. Novel A-ring and B-ring modified combretastatin A-4 (CA-4) analogues endowed with interesting cytotoxic activity. J. Med. Chem. 2008, 51, 6211-6215.
86. Cushman, M.; Nagarathnam, D.; Gopal, D.; Chakraborti, A. K.; Lin, C. M.; Hamel, E. Synthesis and evaluation of stilbene and dihydrostibene derivatives as potential anticancer agents that inhibit tubulin polymerization. J. Med. Chem. 1991, 34, 2579-2588.
87. Lawrence, N. J.; Hepworyh, L. A.; Rennison, D.; McGown, A. T.; Hadfield, J. A. Synthesis and anticancer activity of fluorinated analogues of combretastatin A-4. J. Fluor. Chem. 2003, 123, 101-108.
88. Pettit, G. R.; Rhodes, M. R.; Herald, D. L.; Hamel, E.; Schmidt, J. M.; Pettit, R. K. Antineoplastic Agents. 445. Synthesis and Evaluation of Structural Modifications of (Z)- and (E)-Combretastatin A-4. J. Med. Chem. 2005, 48, 4087-4089.
89. Pinney, K. G.; Mejia, M. P.; Villalobos, V. M.; Rosenquist, B. E.; Pettit, G. R.; Verdier-Pinard, P.; Hamel, E. Synthesis and biological evaluation of aryl azide derivatives of combretastatin a-4 as molecular probes for tubulin. Bioorg. Med. Chem. 2000, 8, 2417-2425.
90. Ohsumi, K.; Nakagawa, R.; Fukuda, Y.; Hatanaka, T.; Morinaga, Y.; Nihei, Y.; Ohishi, K.; Suga, Y.; Akiyama, Y.; Tsuji, T. Novel combretastatin analogues effective against murine solid tumors: design and structure−activity relationships. J. Med. Chem. 1998, 41, 3022-3032.
91. Kong, Y.; Grembecka, J.; Edler, M. C.; Hamel, E.; Mooberry, S. L.; Sabat, M.; Rieger, J.; Brown, M. L. Structure-based discovery of a boronic acid bioisostere of combretastatin A-4. Chem. Biol. 2005, 12, 1007-1014.
92. Monk, K. A.; Siles, R.; Hadimani, M. B.; Mugabe, B. E.; Ackley, J. F.; Studerus, S. W.; Edvardsen, K.; Trawick, M. L.; Garner, C. M.; Rhodes, M. R.; Pettit, G. R.; Pinney, K. G. Design, synthesis, and biological evaluation of combretastatin nitrogen-containing derivatives as inhibitors of tubulin assembly and vascular disrupting agents. Bioorg. Med. Chem. 2006, 14, 3231-3244.
93. Hatanaka, T.; Fujita, K.; Ohsumi, K.; Nakagawa, R.; Fukuda, Y.; Nihei, Y.; Suga, Y.; Akiyama, Y.; Tsuji, T. Novel B-ring modified combretastastin analogues: syntheses and antineoplastic activity. Bioorg. Med. Chem. 1998, 8, 3371-3374.
94. Romagnoli, R.; Baraldi, P. G.; Jung, M. K.; Iaconinoto, M. A.; Carrion, M. D.; Remusat, V.; Preti, D.; Tabrizi, .M. A.; Francesca, F.; Clercq, E. D.; Balzarini, J.; Hamel, E. Synthesis and preliminary biological evaluation of new anti-tubulin agents containing different benzoheterocycles. Bioorg. Med. Chem. Lett. 2005, 15, 4048-4052.
95. Romagnoli, R.; Baraldi, P. G.; Pavani, M. G.; Tabrizi, M. A.; Preti, D.; Fruttarolo, F.; Piccagli, L.; Jung, M. K.; Hamel, E.; Borgatti, M.; Gambari, R. Synthesis and bological evaluation of 2-amino-3-(3‘,4‘,5‘-trimethoxybenzoyl)-5-aryl thiophenes as a new class of potent antitubulin agents. J. Med. Chem. 2006, 49, 3906-3915.
96. Romagnoli, R.; Baraldi, P. G.; Remusat, V.; Carrion, M. D.; Cara, C. L.; Preti, D.; Fruttarolo, F.; Pavani, M. G.; Tabrizi, M. A.; Tolomeo, M.; Grimaudo, S.; Balzarini, J.; Jordan, M. A.; Hamel, E. Synthesis and biological evaluation of 2-(3‘,4‘,5‘-trimethoxybenzoyl)-3-amino 5-aryl thiophenes as a new class of tubulin inhibitors. J. Med. Chem. 2006, 49, 6425-6428.
97. Maya, A. B. S.; Pérez-Melero, C.; Mateo, C.; Alonso, D.; Fernández, J. L. Gajate, C.; Mollinedo, F.; Peláez, R.; Caballero, E.; Medarde, M. Further naphthylcombretastatins. An investigation on the role of the naphthalene moiety. J. Med. Chem. 2005, 48, 556-568.
98. Simoni, D.; Romagnoli, R.; Baruchello, R.; Rondanin, R.; Rizzi, M.; Pavani, M. G.; Alloatti, D.; Giannini, G.; Marcellini, M.; Riccioni, T.; Castorina, M.; Guglielmi, M. B.; Bucci, F.; Carminati, P.; Pisano, C. Novel combretastatin analogues endowed with antitumor activity. J. Med. Chem. 2006, 49, 3134-3152.
99. Reddy, G. R.; Kuo, C. C.; Tan, U. K.; Coumar, M. S.; Chang, C. Y.; Chiang, Y. K.; Lai, M. J.; Yeh, J. Y.; Wu, S. Y.; Chang, J. Y.; Liou, J. P.; Hsieh. H. P. Synthesis and structure−activity relationships of 2-amino-1-aroylnaphthalene and 2-hydroxy-1-aroylnaphthalenes as potent antitubulin agents. J. Med. Chem. 2008, 51, 8163-8167.
100. Mahboobi, S.; Pongratz, H.; Hufsky, H.; Hockemeyer, J.; Frieser, M.; Lyssenko, A.; Paper, D. H.; Bürgermeister, J.; Böhmer, F. D.; Fiebig, H. H.; Burger, A. M.; Baasner, S.; Beckers, T. Synthetic 2-aroylindole derivatives as a new class of potent tubulin-inhibitory, antimitotic agents. J. Med. Chem. 2001, 44, 4535-4553.
101. Romagnoli, R.; Baraldi, P. G.; Sarkar, T.; Carrion, M.; Cara, C. L.; Lopez, C. C.; Preti, D.; Tabrizi, M. A.; Tolomeo, M.; Grimaudo, S.; Cristina, A. D.; Zonta, N.; Balzarini, J.; Brancale, A.; Hsieh, H. P.; Hamel, E. Synthesis and biological evaluation of 1-methyl-2-(3′,4′,5′-trimethoxybenzoyl)-3-aminoindoles as a new class of antimitotic agents and tubulin inhibitors. J. Med. Chem. 2008, 51, 1464-1468.
102. Beckers, T.; Reissmann, T.; Schmidt, M.; Burger, A. M.; Fiebig, H. H.; Vanhoefer, U.; Pongratz, H.; Hufsky, H.; Hockemeyer, J.; Frieser, M.; Mahboobi, S. 2-Aroylindoles, a novel class of potent, orally active small molecule tubulin inhibitors. Cancer Res. 2002, 62, 3113-3119.
103. Liou, J. P.; Chang, Y. L.; Kuo, F. M.; Chang, C. W.; Tseng, H. Y.; Wang, C. C.; Yang, Y. N.; Chang, J. Y.; Lee, S. J.; Hsieh, H. P. Concise synthesis and structure−Activity relationships of combretastatin A-4 analogues, 1-aroylindoles and 3-aroylindoles, as novel classes of potent antitubulin agents. J. Med. Chem. 2004, 47, 4247-4257.
104. Liou, J. P.; Mahindroo, N.; Chang, C. W.; Guo, F. M.; Lee, S. W. H.; Tan, U. K.; Yeh, T. K.; Kuo, C. C.; Chang, Y. W.; Lu, P. H.; Tung, Y. S.; Lin, K. T.; Chang, J. Y.; Hesih, H. P. Structure-activity relationship studies of 3-aroylindiles as potent antimitotic agents. ChemMedChem. 2006, 1, 1106-1118.
105. Wu, Y. S.; Coumar, M. S.; Chang, J. Y.; Sun, H. Y.; Kuo, F. M.; Kuo, C. C.; Chen, Y. J.; Chang, C. Y.; Hsiao, C. L.; Liou, J. P.; Chen, C. P.; Yao, H. T.; Chiang, Y. K.; Tan, U. K.; Chen, C. T.; Chu, C. Y.; Wu, S. Y.; Yeh, T. K.; Lin, C. Y.; Hsieh, H. P. Synthesis and evaluation of 3-aroylindoles as anticancer agents: metabolite approach. J. Med. Chem. 2009, 52, 4941-4945.
106. Liou, J. P.; Wu, Z. Y.; Kuo, C. C.; Chang, C. Y.; Lu, P. Y.; Chen, C. M.; Hsieh, H. P., Chang, J. Y. Discovery of 4-amino and 4-hydroxy-1-aroylindoles as potent tubulin polymerization inhibitors. J. Med. Chem. 2008, 51, 4351-4355.
107. Liou, J. P.; Wu, C. Y.; Hsieh, H. P.; Chang, C. Y.; Chen, C. M.; Kuo, C. C.; Chang, J. Y. 4- and 5-aroylindoles as novel classes of potent antitubulin agents. J. Med. Chem. 2007, 50, 4548-4552
108. Hewlins, M. J. E.; Jackson, A. H.; Oliveira-Campos, A. M.; Shannon, P. V. R. Synthesis of 8,9,10-trimethoxyellipticine. J. Chem. Soc., Perkin Trans. 1, 1981, 2906-2911.
109. Coowar, D.; Bouissac, J.; Hanbali, M.; Paschaki, M.; Mohier, E.; Luu, B. Effects of Indole Fatty Alcohols on the Differentiation of neural stem cell derived nurospheres. J. Med. Chem., 2004, 47, 6270–6282.
110. Banwell, M. G.; Hamel, E.; Hockless, D. C. R.; Verdier-Pinard, P.; Willis, A. C.; Wong, D. J. 4,5-Diaryl-1H-pyrrole-2-carboxylates as combretastatin A-4/lamellarin T hybrids: Synthesis and evaluation as anti-mitotic and cytotoxic agents. Bioorg. Med. Chem. 2006, 14, 4627-4638.
111. Zhang, H.; Cai, Q.; Ma, D. Amino acid promoted CuI-catalyzed C−N bond formation between aryl halides and amines or N-containing heterocycles. J. Org. Chem., 2005, 70, 5164–5173.
112. Fryatt, T.; Pettersson, H. I.; Gardipee, W. T.; Bray, K. C.; Green, S. J.; Slawin, A. M. Z.; Beall, H. D.; Moody, C. J. Novel quinolinequinone antitumor agents: structure-metabolism studies with NAD(P)H:quinone oxidoreductase (NQO1). Bioorg. Med. Chem. 2004, 12, 1667-1687.
113. Fink, C. A.; Perez, L. B.; Ramsey, T. M.; Yusuff, N.; Versace, R. W.; Batt, D. B.; Sabio, M. L.; Kim, S. 1,4-Disubstituted isoquinilone derivatives as raf-kinase inhibitors useful for the treatment of proliferative diseases. WO2005028444, 2005.
114. Xu, W. S.; Parmigiani, R. B.; Marks, P. A. Histone deacetylase inhibitors: molecular mechanism of action. Oncogene 2007, 26, 5541-5552.
115. Dokmanovic, M.; Clarke, C.; Marks, P. A. Histone deacetylase inhibitors: overview and perspectives. Mol. Cancer Res. 2007, 5, 981-989.
116. Paris, M.; Porcelloni, M.; Binaschi, M.; Fattori, D. Histone deacetylase inhibitors: from bench to clinic. J. Med. Chem. 2008, 51, 1505-1529.
117. De Ruijter, A. J.; Van Gennip, A. H.; Caron, H. N.; Kemp, S.; Van Kuilenburg, A. B. Histone deacetylases (HDACs): characterization of the classical HDAC family. Biochem J. 2003, 370, 737-749.
118. Gallinari, P.; Di Marco, S.; Jones, P.; Pallaoro, M.; Steinkuhler, C. HDACs, histone deacetylation and gene transcription: from molecular biology to cancer therapeutics. Cell Res. 2007, 17, 195-211.
119. Kawaguchi, Y.; Kovacs, J. J.; McLaurin, A.; Vance, J. M.; Ito, A.; Yao, T. P. The deactylase HDAC6 regulates aggresome formation and cell viability in response to misfolded protein stress. Cell. 2003, 115, 727-738.
120. Hideshima, T.; Bradner, J. E.; Wong, J.; Chauhan, D.; Richardson, P.; Schreiber, S. L.; Anderson, K. C. Small-molecule inhibition of proteasome and aggresome function induces synergistic antitumor activity in multiple myeloma. Proc. Natl. Acad. Sci. U.S.A. 2003, 102, 8567-8572.
121. Butler, R.; Bates, G. P. Histone deacetylase inhibitors as therapeutics for polyglutamine disorders. Nat. Rev Neurosci. 2006, 7, 784-796.
122. Kovacs, J. J.; Murphy, P. J.M.; Gaillard, S.; Zhao, X.; Wu, J. T.; Nicchitta, C. V.; Yoshida, M.; Toft, D. O.; Pratt, W. B.; Yao, T. P. HDAC6 regulates Hsp90 acetylation and chaperone-dependent activation of glucocorticoid receptor. Mol. Cell. 2005, 18, 601-607.
123. Finnin, M.S.; Donigian, J. R.; Cohen, A.; Richon, V. M.; Rifkind, R. A.; Marks, P. A.; Breslow, R.; Pavletich, N. P. Structure of a histone deacetylase homologue bound to TSA and SAHA inhibitors. Nature 1999, 401, 188-193.
124. Miller, T. A.; Witter, D. J.; Belvedre, S. Histone deacetylase inhibitors. J. Med. Chem. 2003, 46, 5097-5116.
125. Vannini, A.; Volpari, C.; Filocamo, G.; Caroli Casavola, E.; Brunetti, M.; Renzoni, D.; Chakravarty, P.; Paolini, C.; De Francesco, R.; Gallinari, P.; Steinkuhler, C.; Di Marco, S. Crystal structure of a eukaryotic zinc-dependent histone deacetylase, human HDAC8, complexed with a hydroxamic acid inhibitors. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 15064-15069.
126. Wang, D.F.; Helquist, P.; Wiech, N. L.; Wiest, O. Toward selective histone deacetylase inhibitors design: homology modeling, docking studies, and molecular dynamic simulations of human class I histone deacetylases. J. Med. Chem. 2006, 49, 6936-6947.

127. Dokmanovic M.; Marks, P. A. Prospects: histone deacetylase inhibitors. J. Cell. Biochem. 2005, 96, 293-304.
128. Price, S.; Dyke, H. J. Histone deacetylase inhibitors: an analysis of recent patenting activity. Expert Opin. Ther. Pat. 2007, 17, 745-765.
129. Glaser, K. B. HDAC inhibitors: clinical update and mechanism-based potential. Biochem. Pharmacol. 2007, 74, 659-671.
130. Marks, P. A. Discovery and development of SAHA as an anticancer agent. Oncogene 2007, 26, 1351-1356.
131. Belvedere, S.; Witter, D. J.; Yan, Y.; Secrist, P.; Richon, V.; Miller, T. A. Aminosuberoyl hydroxamic acids (AHSAs): a potent new class of HDAC inhibitors. Bioorg. Med. Chem. Lett. 2007, 17, 3969-3971.
132. (a) Belvedere, S.; Methot, J. L.; Miller, T. A.; Witter, D. J.; Yan, J. Histone Deacetylases Inhibitors. WO2006017215, 2006. (b) Belvedere, S.; Hamblett, C. L.; Miller, T. A.; Witter, D. J.; Yan, J. Histone Deacetylases Inhibitors. WO2006017216, 2006. (c) Belvedere, S.; Hamblett, C. L..; Miller, T. A.; Witter, D. J.; Yan, J. Histone Deacetylases Inhibitors. WO2006026260, 2006.
133. Miller, T. A.; Witter, D. J.; Belvedere, S. Diamine and Iminodiacetic Acid Hydroxamic Acid Derivatives. WO2005053610, 2005.
134. Dai, Y.; Guo, Y.; Curtin, M. L.; Li, J.; Pease, L. J.; Guo, J.; Marcotte, P. A.; Glaser, K. B.; Davidsen, S. K.; Michaelides, M. R.; A novel series of histone deacetylase inhibitors incorporating heteroaromatic ring systems as connection units. Bioorg. Med. Chem. Lett. 2003, 12, 3817-3820.
135. Cho, J. W.; Lim, S. C.; Maeng, C. Y.; Hwang, S. G.; Bae, S. J.; Kim, E. A. Oxazole Hydroxamic Acid Derivatives and Use Thereof. WO2006075888, 2006.
136. Kozikowski, A. P.; Dritschilo, A.; Jung, M.; Petukhov, P.; Chen, B. Histone Deacetylase Inhibitors and Methods of Use Thereof. WO2005007091, 2005.
137. Scopes, D. I. C. Substituted Phenylurea Derivatives as HDAC Inhibitors. WO2004067480, 2004.
138. Marson, C. M.; Savy, P.; Rioja, A. S.; Mahadevan, T.; Mikol, C.; Veerupillai, A.; Nsubuga, E.; Chahwan, A.; Joel, S. P. Aromatic sulfide inhibitors of histone deacetylase based on arylsulfinyl-2,4-hexadienoic acid hydroxamides. J. Med. Chem. 2006, 49, 800-805.
139. Mai, A.; Massa, S.; Rotili, D.; Simeoni, S.; Ragno, R.; Botta, G.; Nebbioso, A.; Miceli, M.; Altucci, L.; Brosch, G. Synthesis and biological properties of novel, uracil-containing histone deacetylase inhibitors. J. Med. Chem. 2006, 49, 6046-6056.
140. Glenn, M. P.; Kahnberg, P.; Boyle, G. M.; Hansford, K. A.; Hans, D.; martyn, A. C.; Parsons, P. G.; Fairlie, D. P. Antiproliferative and phenotype-transforming antitumor agents derived from cysteine. J. Med. Chem. 2004, 47, 2984-2994.
141. Rossi, C.; Porcelloni, M.; D’Andrea, P.; Fattori, D.; Marastoni, E. Hydroxamates as Histone Deacetylase Inhibitors and Pharmaceutical Formulations Containing Them. WO2006097460, 2006.
142. Guidi, A.; Dimoulas, T.; Giannotti, D.; Harmat, N. N-Hydroxamides ω–Substituted with Tricyclic Groups as Histone Deacetylase Inhibitors, Their Preparation and Use in Pharmaceutical Formulations. WO2006097449, 2006.
143. Finn, P. W.; Bandara, M.; Butcher, C,; Finn, A.; Hollinshead, R.; Khan, N.; Law, N.; Murthy, S.; Romero, R.; Watkins, C.; Andriannov, V.; Bokaldere, R. M.; Dikovska, K.; Gailite, V.; Loza, E.; Piskunova, I.; Starchenkov, I.; Vorona, M.; Kalvinsh, I. Novel sulfonamide derivatives as inhibitiors of histone deacetylase. Helv. Chim. Acta 2005, 88, 1630-1657.
144. (a) Kim, D.-K.; Lee, J. Y.; Kim, J.-S.; Ryu, J.-H.; Choi, J.-Y.; Lee,J. W.; Im, G.-J.; Kim, T.-K.; Seo, J. W.; Park, H.-J.; Yoo, J.; Park, J.-H.; Kim, T.-Y.; Bang, Y.-J. Synthesis and biological evaluation of 3-(4-substituted-phenyl)-N-hydroxy-2-propenamides, a new class of histone deacetylase inhibitors. J. Med. Chem. 2003, 46, 5745-5751. (b) Kim, D.-K.; Lee, J. Y.; Lee, N. K.; Kim, J.-S.; Lee, J. W.; Lee, S. H.; Choi, J.-Y.; Ryu, J.-H.; Kim, N. H.; Im, G.-J.; Kim, T.-K.; Seo, J, W.; Bang, Y.-J. α,β-Unsaturated Hydroxamic
Acid Derivatives and Their Use as Histone Deacetylase Inhiitors. WO200387066, 2003.
145. Urano, Y.; Satoh, S.; Ishibashi, N.; Kamijo, K. Hydroxamic Acid Derivative as Histone Deacetylase (HDAC) Inhibitors. WO2004063169, 2004.
146. Shinji, C.; Maeda, S.; Imai, K.; Yoshida, M.; Hashimoto, Y.; Miyachi, H. Design, synthesis and evaluation of cyclic-selective histone deacetylase (HDAC) inhibitors. Bioorg. Med. Chem. 2006, 8, 7625-7651.
147. (a)Mai, A.; Massa, S.; Cerbara, I.; Valente, S.; Ragno, R.; Bottoni, P.; Scatena, R.; Loidl, P.; Brosch, G. 3-(4-Aroyl-1-methyl-1H-2-pyrrolyl)-N-hydroxy-2-propenamides as a new class of synthetic histone deacetylase inhibitors. 2. Effect of pyrrole-C2 and/or –C4 substitutions on biological activity. J. Med. Chem. 2004, 47, 1098-1109. (b) Rango, R.; Mai, A.; Massa, S.; Cerbara, I.; Valente, S.; Bottoni, P.; Scatena, R.; Jesacher, F.; Loidl, P.; Brosch, G. 3-(4-Aroyl-1-methyl-1H-pyrrol-2-yl)-N-hydroxy-2-propenamides as a new class of synthetic histone deacetylase inhibitors. 3. Discovery of novel lead compounds through structure-based drug design and docking studies. J. Med. Chem. 2004, 47, 1351-1359. (c) Mai, A.; Massa, S.;Pezzi, R.; Simeoni, S.; Rotili, D.; Nebbioso, A.; Scoganamiglio, A. Altucci, L.; Loidl, P.; Brosh, G. Class II (IIa)-selective histone deacetylase inhibitors. 1. Synthesis and biological evaluation of novel (aryloxopropenyl)pyrrolyl hydroxamides. J. Med. Chem. 2003, 46, 4826-4829.
148. Minucci, S.; Pelicci, P. G.; Mai, A.; Ballarini, M.; Gargiulo, G.; Massa, S. New Histone Deacetylase Inhibitors. WO2006037761, 2006.
149. Lee, K. C.; Sun, E. T. Imidazole[1,2-A]pyridine Derivatives: Preparation and Pharmaceutical Applications. WO2006101455, 2006.
150. (a) Groosmann, A.; Herting, F.; Koerner, M.; Kuenkele, K.-P.; Limberg, A.; Mundigl, O.; Tibes, U. Enantiomers of Thiophene Hydroxamic Acid Derivatives and Their Use as HDAC Inhibitors. WO200405499, 2004. (b) Fertig, G.; Herting, F.; Koerner, M.; Kubbies, M.; Limberg, A.; Reiff, U.; Tibes, U. Hydroxamates, Their Manufacture and Use as Pharmaceutical Agents. WO200512119, 2005. (c) Fertig, G.; Herting, F.; Koerner, M.; Kubbies, M.; Limberg, A.; Reiff, U.; Tibes, U. Thiophene Hydroxamic Acid Derivatives and Their Use as HDAC Inhibitors. WO2005121120, 2005. (d) Ferting, G.; Herting, F.; Koerner, M.; Kubbies, M.; Limberg, A.; Reiff, U.; Tibes, U. Thiophene Derivatives, Their Manufacture and Use As Pharmaceutical Agents. WO2005121134, 2005.
151. (a) Archer, J. A.; Bordogna, W.; Bull, R. J.; Clark, D. E.; Dyke, H. J.; Gill, M.; Harris, N. V.; Van Den Heuvel, M.; Price, S. Substituted Thienyl-hydroxamic Acids as Histone Deacetylase Inhibitors. WO2004013130, 2004. (b) Bordogna, W.; Sutton, J. M.; Hynd, G.; Dyke, H. J.; Price, S.; Harris, N. V.; Gill, M. I. A. Thiazolyl-hydroxamic Acids and Thiadiazolyl-hydroxamic Acids, and Use Thereof for Treating Diseases Associated with Histone Deacetylase Enzymatic Activity. WO2005075469, 2005. (c) Dyke, H. J.; Price, S.; Van Den Heuvel, M.; Sutton, J. M; MacKenzie, R. E.; Heald, R. E. Substituted Thienyl-hydroxamic Acids Having Histone Deacetylase Activity. WO2005014588, 2005.
152. Stunkel, W.; Wang, H.; Yin, Z. Biaryl Linked Hydroxamates: Preparation and Pharmaceutical Applications. WO2005040161, 2005.

153. Uesato, S.; Nagaoka, Y.; Yamori, T. N-Hydroxycarboxamide Derivative. WO2003070691, 2003.
154. Chakravarty, P. K.; Kuo, H.; Matthews, J. M.; Meinke, P. T. Inhibitors of Histone Deacetylase. WO2006017214, 2006.
155. Anandan, S. K.; Xiao, X.-Y.; Ward, J. S.; Patel, D. V. Fused Heterocyclic Compounds Useful as Inhibitors of Histone Deacetylase. WO2006088949, 2006.
156. Miller, T. A.; Witter, D. J.; Belvedere, S. Thiophene and Benzothiophene Hydroxamic Acid Derivatives. WO2005034880, 2005.
157. Bressi, J. C.; Gangloff, A. R.; Jennings, A. J. Histone Deacetylase Inhibitors. WO2005066151, 2005.
158. Suzuki, T.; Kouketsu, A.; Matsuura, A.; Kohara, A.; Ninomiya, S.-I.; Kohda, K.; Miyata, N. Thiol-based SAHA analogues as potent histone deacetylase inhibitors. Bioorg. Med. Chem. Lett. 2004, 14, 3313-3317.
159. Chen, B.; Petukhov, P. A.; Jung, M.; Velena, A.; Eliseeva, E.; Dritschilo, A.; Kozikowski, A. P. Chemistry and biology of mercaptoacetamides as novel histone deacetylase inhibitors. Bioorg. Med. Chem. Lett. 2005, 15, 1389-1392.
160. Gu, W.; Nusinzon, I.; Smith, R. D.; Horvath, C. M.; Silverman, R. B. Carbonyl- and sulfur-containing analogs of suberoylanilide hydroxamic acid: potent inhibition of histone deacetylases. Bioorg. Med. Chem. 2006, 14, 3320-3329.
161. Vaisburg, A.; Bernstein, N.; Frechette, S.; Allan, M.; Abou-Khalil, E.; Leit, S.; Moradei, O.; Bouchain, G.; Wang, J.; Woo, S. H.; Fournel, M.; Yan, P. T.; Trachy-Bourget, M. C.; Kalita, A.; Beaulieu, C.; Li,Z.; Mac Leod, A. R.; Besterman, J. M.; Delorme, D. (2-Amino-phenyl)-amides of ω–substituted alkanoic acids as new histone deacetylase inhibitors. Bioorg. Med. Chem. Lett. 2004, 14, 283-287.

162. Moradei, O.; Leit, S.; Zhou, N.; Frechette, S.; Paquin, I.; Raeppel, S.; Gaudette, F.; Bouchain, G.; Woo, S. H.; Vaisburg, A.; Fournel, M.; Kalita, A.; Lu, A.; Trachy-Bourget, M. C.; Yan, P. T.; Liu, J.; Li, Z.; Rahil, J.; MacLeod, A. R.; Besterman, J. M.; Delorme, D. Substituted N-(2-amino-phenyl)-benzamides, (E)-N-(2-aminophenyl)-acrylamides and their analogues: novel classes of histone deacetylase inhibitors. Bioorg. Med. Chem. Lett. 2006, 16, 4048-4052.
163. Nagaoka, Y.; Maeda, T.; Kawai, Y.; Nakashima, D.; Oikawa, T.; Shimoke, K.; Ikeuchi, T.; Kuwajima, H.; Uesato, S. Synthesis and cancer antiperliferative activity of new histone deacetylase inhibitors: hydrophilic hydroxamates and 2-aminobenzamide-containing derivatives. Eur. J. Med. Chem. 2006, 41, 697-708.
164. Siliphaivanh, P.; Harrington, P.; Witter, D. J.; Otte, K.; Tempest, P.; Kattar, S.; Kral, A. M.; Fleming, J. C.; Deshmukh, S. V.; Harsch, A. Secrist, P. J.; Miller, T. A. Design of novel histone deacetylase inhibitors. Bioorg. Med. Chem. Lett. 2007, 17, 4619-4624.
165. (a) Frey, R. R.; Wada, C. K.; Garland, R. B.; Curtin, M. L.; Michaelides, M. R.; Li, J.; Pease, L. J.; Glaser, K. B.; Marcotte, P. A.; Bouska, J. J.; Murphy, S. S.; DAvidsen, S. K. Trifluoromethyl ketones as inhibitors of histone deacetylase. Bioorg. Med. Chem. Lett. 2002, 12, 3443-3447. (b) Wada, K. C.; Frey, R. R.; Ji, Z.; Curtin, M. L.; Garland, R. B.; Holma, J. H.; Li, J. Pease, L. J.; Guo, J.; Glaser, K. B.; Marcotte, P. A.; Richardson, P. L.; Murphy, S. S.; Bouska, J. J.; Tapang, P.; Magoc, T. J.; Albert, D. H.; Davidsen, S. K.; Michaelides, M. R. α-Keto amides as inhibitors of histone deacetylase Bioorg. Med. Chem. Lett. 2003, 13, 3331-3335. (c) VAsudevan, A.; Ji, Z.; Frey, R. R.; Wada, K. C.; Steinman, D.; Heyman, H. R.; Guo, Y.; Curtin, M. L.; Guo, J.; Li, J.; Pease, L.; Glaser, K. B.; Marcotte, P. A.; Bouska, J. J.; Albert, D. H.; Davidsen, S. K.; Michaelides, M. R. Heterocyclic ketones as inhibitors of histone deacetylase. Bioorg. Med. Chem. Lett. 2003, 13, 3909-3913.
166. (a) Jones, P.; Altamura, S.; Chakravarty, P. K.; Cecchetti, O.; De Francesco, R.; Gallinari, P.; Ingenito, R.; Meinke, P. T.; Petrocchi, A.; Rowley, M.; Scarpeli, R.; Serafini, S.; Steinkuhler, C. A series of novel, potent and selective histone deacetylase inhibitors. Bioorg. Med. Chem. Lett. 2006, 16, 5948-5952. (b) Chakravarty, P. K.; Colletti, S. L.; Ingenito, R.; Jones, P.; Meinke, P. T.; Muraglia, E.; Peterocchi, A.; Rowley, M.; Scarpelli, R.; Steinkuhler, C. Amides Derivatives as Inhibitors of Histone Deacetylase. WO2006005941, 2006. (c) Chakravarty, P. K.; Colletti, S. L.; Ingenito, R.; Jones, P.; Meinke, P. T.; Petrocchi, A.; Steinkuhler, C. Amides Derivatives as Inhibitors of Histone Deacetylase. WO2006005955, 2006.
167. Hu, E.; Dul, E.; Sung, C.-M.; Chen, Z.; Kirkpatrick, R.; Zhang, G.-F.; Johanson, K.; Liu, R.; Lago, A.; Hofmann, G.; Macarron, R.; de los Frailes, M.; Perez, P.; Krawiec, J.; Winkler, J.; Jaye, M. Identification of novel isoform-selective inhibitors within class I histone deacetylases. J. Pharmacol. Exp. Ther. 2003, 307, 720-728.
168. Oku, N.; Nagai, K.; Shindoh, N.; Terada, Y.; van Soest, R. W. M.; Matsunaga, S.; Fusetani, N. Three new cyclostellettamines, which inhibit histone deacetylase from a marine sponge of the genus Xestospongia. Bioorg. Med. Chem. Lett. 2004, 14, 2617-2620.

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系統識別號 U0007-1704200714512810
論文名稱(中文) 去乙醯幾丁聚醣/膠原蛋白組織再生膜片之活體評估
論文名稱(英文) Collagen-Chitosan Composite Barrier For Guided Tissue Regeneration
校院名稱 臺北醫學大學
系所名稱(中) 牙醫學系碩博士班
系所名稱(英) Graduate School of Dentistry
學年度 91
學期 2
出版年 92
研究生(中文) 許文祥
學號 M88040134
學位類別 碩士
語文別 中文
口試日期
論文頁數 84頁
口試委員 指導教授-李勝揚
指導教授-王敦正
關鍵字(中) 去乙醯幾丁聚醣
膠原蛋白
引導組織再生術
牙堊質再生高度
齒槽骨再生高度
關鍵字(英) chitosan
collagen
guided tissue regeneration
cementum height
alveolar bone regeneration
學科別分類
中文摘要 本研究之目的在評估與工研院生醫中心合作發展出可吸收性去乙醯幾丁聚醣/膠原蛋白複合之組織導引再生膜片,並選擇與可吸收性之BioMend Extend™ 及Peri-Aid® 膠原蛋白膜片,和GORE-TEX® OSSEOQUEST 合成高分子膜片,及不可吸收之GORE-TEX® e-PTFE合成高分子膜片,等其他四種市售商品材料共同對照評估,應用8隻年齡為12個月的雄性小獵犬(Beagle dogs) ,均分4組 (分別為7天、14天、28天、3個月),為動物活體評估模式,在實驗犬之左、右下顎第一、二小臼齒及大臼齒的頰側區製造骨缺損後,分別植入組織導引再生膜片,依實驗設定時間將小獵犬犧牲,取下缺損區骨頭,以光學顯微鏡觀察量測其牙堊質再生高度與齒槽骨再生高度之變化,以探討其組織再生模式及評估新膜片在臨床應用之適用性。結果顯示,去乙醯幾丁聚醣/膠原蛋白膜片在手術後第4週牙堊質再生高度平均為0.9 mm,效果上與市售膠原蛋白膜片(2.6 mm)相近。術後第3個月時,牙堊質再生高度可達2.6 mm,明顯優於e-PTFE膜片 (2.32 mm);在新生骨之生成方面,膜片植入後三個月後,可發現槽骨再生高度平均可達1.16 mm,與市售可吸收性膜片(1.0 mm)類似,但明顯優於e-PTFE膜片 (0.74 mm)。綜合以上觀察,在防止上皮細胞向牙根尖部生長,及促使牙周結締組織生長於牙根表面上,所開發之去乙醯幾丁聚醣/膠原蛋白膜片較市售膜片有相似或更佳之功能,同時在癒合初期能促進血塊凝結與減少感染的機率,而有助於傷口之穩定。 關鍵字:去乙醯幾丁聚醣,膠原蛋白,引導組織再生術,牙堊質再生高度,齒槽骨再生高度
英文摘要 Abstract This in vivo study was to examine the historical changes of implanted novel chitosan/collagen composite barrier for confirming the clinical feasibility. Four other commercial GTR (Guided Tissue Regeneration) membranes were chosen for comparison. Among the resorbable GTR membranes, BioMend Extend™ and Peri-Aid® are collagen base, and GORE-TEX® OSSEOQUEST is synthesized membrane, while GORE-TEX® e-PTFE (Expanded polytetrafluoroethylene) is synthesized but non-resorbable. Beagle dogs were used as animal model. Buccal mucoperiosteal flaps were reflected in the bilateral mandibular premolar and molar areas. Buccal alveolar bone was reduced on 1st、2nd premolar and molar to a level 5 mm apical to the cemento-enemel junction (CEJ). Root surface was denuded of periodontal ligament and cementum, and notches were placed at the bone level of each root. The tested GTR barriers were implanted in critical bone defect areas. Flaps were coronally positioned and sutured. Two beagle dogs were sacrificed each time as the designed time period after surgery. Histological and histometirc evaluation at 7 days、14 days、28 days、3 months were performed post-operatively to determine the healing response of each treatment modality. Both the cementum height and bone height were measured as the index of tissue occlusion effect. Like all resorbable GTR membrane, the chitosan/collagen composite barrier enhanced the cementum regeneration of 1.16 mm averagely after 28-day implantation. After 3 months, average cementum height of 2.6 mm was observed for chitosan/collagen composite barrier group. On the contrast, cementum height of 0.9 mm was observed in e-PTFE group. Meanwhile bone regeneration also observed and bone height was measured average 1.0 mm for all test membrane group except the control group without GTR membrane implanted reveal no bone formation. In our study, inhibiting epithelial migration and encouraging formation of new connective tissue attachment to root surface evidenced positive results of chitosan/collagen composite barrier placement. It also promoted blood clot aggregation and maturation in early wound healing process and decreased wound infection. Key word:chitosan, collagen, guided tissue regeneration, cementum height, alveolar bone regeneration.
論文目次 目 錄 私立台北醫學大學口腔復健醫學研究所…………………………I 誌謝…………………………………………………………………II 論文摘要……………………………………………………………III Abstract……………………………………………………………IV 論文口試委員簽名…………………………………………………V 碩士論文繕印同意書………………………………………………VI 目錄…………………………………………………………………8 第一章 動機與序論……………………………………………… 11 第一節 研究動機……………………………………………… 12 第二節 研究假設……………………………………………… 13 第三節 研究目的……………………………………………… 13 第四節 名詞界定……………………………………………… 14 第二章 文獻回顧………………………………………………… 19 第一節 引導組織再生術……………………………………… 20 第二節 膠原蛋白膜片………………………………………… 22 第三節 去乙醯幾丁聚醣……………………………………… 24 第三章 研究材料與方法………………………………………… 27 第一節 實驗材料……………………………………………… 28 第二節 動物選取和處理……………………………………… 33 第三節 實驗步驟……………………………………………… 34 第四章 實驗結果………………………………………………… 37 第一節 外觀觀察……………………………………………… 38 第二節 組織學觀察…………………………………………… 39 第五章 討論……………………………………………………… 43 第六章 參考文獻………………………………………………… 53
參考文獻 1.Glossary of Periodontal Term. 3th Edition. 1992 2.Alan S, James L. History. 1994:43 3.Muzzzarelli R A A, Jeuniaux C, Gooday G W. Chitin in nature and Technology, Plenum Press, New York, 1986 4.Aiba S, Minoura N, Taguchi C, et al. Covalent immobilization of chitosan derivatives onto polymeric film surfaces with the use of photosensitive hetero-bifunctional crosslinking reagent. Biomaterials. 1987;8:481-488 5.Peterson LJ, Ellis E, Hupp JR, Tucker MR, et al. Comtemporary Oral and Maxillofacial Surgery. 2nd. 1993 6.Kon S, Ruben MP, Bloon AA, et al. Regeneration of Periodontal Ligament Using Resorbable and Noresorabable Membrane: Clinical, Histological, and Histometric Study in Dog. Int J Periodontics Restorative Dent. 1991; 11:59-71. 7.Pitura S, Tal H, Soldinger M, et al. Collagen membranes prevent apical migration of epithelium during periodontal wound healing. Journal of Periodontal Research. 1987; 22: 331-333. 8.Blumental NM. The Use of Collagen Membranes to Guide Regeneration of New Connective Tissue Attachment on Dogs. J Periodontol. 1988;59:830-836. 9.Pitura S, Tal H, Soldinger M, et al. Partial Regeneration of Periodontal Tissues Using Collagen Barriers. J Periodontol. 1988; 59:380-386. 10.Aukhil I, Simpson DM, Schaberg TV. An experimental study of new attachment procedure in beagle dogs. Journal of Periodontal Research. 1983;18: 643-654. 11.Card SJ, Caffesse RG, Smith BA, et al. New Attachment Following the Use of a Resorbable Membrane in the Treatment of Periodontal in Dogs. The International Journal of Periodontics and Restorative Dentistry. 1989;9:59-69. 12.Caffesse RG, Nasjleti CE, Morrison EC, et al. Guided Tissue Regeneration: Comparison of Bioabsorable and NonBioabsorbable Membranes. Histologic and Histometirc Study in Dogs. J Periodontal. 1994;65:583-591. 13.Zellin G, Gritli-Linde A, Linde A. Healing of mandibular defect with different biodegradable and non- biodegradable membranes: an experimental study in rats. Biomaterials. 1995; 16:601-609. 14.Galgut P, Pitrola R, Waite I, et al. Histological evaluation of biodegradable and non-degradable membranes placed transcutaneously in rats. J Clin Periodotol. 1991;18:581-586. 15.Hyder PR, Dowell P, Singh G., et al.. Freeze-Dired, Cross-linked Bovine Type 1 Collagen: Analysis of Properties. J Periodontol. 1992;63; 3:182-186. 16.Chaput C, Guirguis S, Leroux JC, et al. Characterization of thermosensitive chitosan gels for the sustained delivery of drugs. International Journal of Pharmaceutics. 2000;203:89-98. 17.Francis Suh JK, Howard WT. Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: a review. Matthew Biomaterials. 2000; 21:2589-2598. 18.Pack YJ, Lee YM, Park SN, et al. Platelet derived growth factor releasing chitosan sponge for periodontal bone regeneration. Biomaterials. 2000;21:153-159. 19.Aiba S, Minoura N, Taguchi C, et al. Covalent immobilization of chitosan derivatives onto polymeric film surfaces with the use of photosensitive hetero-bifunctional crosslinking reagent. Biomaterials. 1987;8:481-488 20.Postlethwaite AE, Seyer JM, Kang AH. Chemotactic attraction of human fibroblast to type I, II, III collagens and collagen derived peptides. Proc Natl Acad Sci (USA) 1978:75;817-875 21.Saleman E, Biollogy, Biotechnology and Biocompatibility of collagen. Biocompatibilty of Tissue Analogs, 1 st ed. Boca Raton , FL:CRC Press, Inc; 1985;27 22.Locci P, Calvitti M, Belcastro S, et al. Phenotype expression og gingival fibroblast cultured on membrane used in guided tissue regeneration. J. Periodontal. 1997;68 :857-863 23.Schlegel AK, Mohler H, Busch F, et al. Preclinical and clinical studies of a collagen membrane (Bio-Gide). Biolmaterials 1997;18:535-538 24.Johns LP, Merritt K, Agrarwal S, et al. Immunogenicity of a bovine collagen membrane in guided tissue regeneration. J Dent Res. 1992;71:298 25.Makajima M, Athsumi K, Kifune K, et al. Chitin is an effective material for suture. Jan. J. Surg. 1986;16:418-424 26.Yannas IV, Bruk JF, Huuang C, et al. Correlation of in vivo collagen degradation rate with in vitro measurements. J Biomed Master Res. 1975;9:623-628 27.Quinn KJ, Courtne JM, Evans JH, et al. Principle of burn dressing. Biomaterials. 1985;6:369-377 28.Solum NO. Platelet aggregation during fibrin polymerization. Scand. J. clin. Lab. Invest. 1966:18;577-582 29.Grinnell F, Feld M, Minter D. Fibroblast adhesion to fibrinogen substrata, Requirement for cold insoluble globulin (plasma fibronectin). Cell. 1980;19:517-525 30.Knox P, Crooks S, Rimmer C, et al. Role of fibronectin in the migration of fibroblast into plasma clots. J. Cell. Bio. 1986;102:2318-2323 31.Fine A, Goldstein R H. The effect of transforming growth factor B on cell proliferation and collagen formation by lung fibroblasts. J. Biol. Chem. 1987;262: 3897-3902 32.Schmitz JP, Hollinger JO. The critical size defect as an experimental model for craniomandibulofacial nonunions. Clin Orthop. 1986; 205: 229-308 33.Alberius P, Dahin C, Linde A. Role of osteopromotion in experimental bone grafting to the skull: A study in adult rats using a membrane technique. J Oral Maxillofac Surg. 1992; 50: 829-834 34.Linde A, Thoren C, Dahlin C, et al. Creation of new bone by an osteopromotive membrane technique. An experimental study in rats. J Oral Maxillfac Surg. 1993;51:892-897. 35.Gottlow J, Nyman S, Karring, et al. Healing following citric acid conditioning of roots implanted into bone gingival connective tissue. J Periodontal Res. 1984;19:214-22 36.Karring T, Nyman S, Lindhe J, et al. Healing following implantation of periodontitis affected roots into bone tissue. J clin. Periodontal. 1980;7:96-105 37.Nyman S, Karring T , Lindhe J, Planten S, et al. Healing following implantation of periodontitis affected roots into gingival connective tissue. J clin. Periodontal. 1980;7:394-401 38.Karring T, Isidor F, Nyman S, Lindhe J, et al. New attachment formation on teeth with a reduced but healthy periodontal ligament. J clin. Periodontal. 1985;12:51-60 39.Nyman S, Gottlow J, Karring T, Lindhe J, et al. The regenerative potential of the periodontal ligament. An experimental study in the monkey. J clin. Periodontal. 1982;9:257-265 40.Gottlow J, Nyman S, Karring T, Lindhe J, et al. New attachment formation as the result of controlled tissue periodontium by guided tissue regeneration. J clin. Periodontal. 1984; 11:494-503 41.Becker W, Becker B, Prichard R, et al. Root isolation for new attachment procedure: a surgical and suture method. Three case reports. J Periodontal. 1987;58: 819-826 Stahl S, Forum S, Tarnow D, et al. Human histological responses to guided tissue regenerative techniques in intrabony lesions. Case reports on 9 sites. J. clin. Periodontal. 1990; 17:191-198

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系統識別號 U0007-1704200714541602
論文名稱(中文) 生物檢體中脂蛋白脂?活性測定之螢光高效能液相層析法的開發
論文名稱(英文) Determination of lipoprotein lipase activity in biological samples by high performance liquid chromatography with fluorescence detection
校院名稱 臺北醫學大學
系所名稱(中) 藥學研究所
系所名稱(英) Graduate Institute of Pharmacy
學年度 93
學期 2
出版年 94
研究生(中文) 周郁晴
學號 m301092007
學位類別 碩士
語文別 中文
口試日期
論文頁數 86頁
口試委員 指導教授-李仁愛
關鍵字(中) 脂蛋白脂?
螢光高效能液相層析法
乙醯肝素
分析法
脂肪酸
脂蛋白
糖尿病大鼠
關鍵字(英) lipoprotein lipase
HPLC
NBD-PZ
STZ-induced diabetic rats
學科別分類
中文摘要 脂蛋白脂?(Lipoprotein lipase, LPL)是由組織中的薄壁細胞所合成,以硫酸乙醯肝素黏附於微血管內皮細胞上,主要負責分解血漿中之三酸甘油酯。LPL為脂蛋白代謝中不可或缺的一環,其活性高低與糖尿病、心血管疾病、血脂異常…等疾病有關。 本研究係發展一套螢光高效能液相層析法,使用螢光衍生化試劑4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ),將LPL與受質作用所產生之游離脂肪酸衍生化後分析,藉此定量LPL的活性。透過本分析法,檢品不須經過萃取步驟,並能完全分離干擾物質、衍生化之脂肪酸、和參與反應的受質。研究選用triolein為受質,其飽和濃度為10 mM,我們發現酵素與受質的作用須選用適當的乳化劑,透過血清白蛋白媒介,才能有效進行,實驗結果顯示1%的Gum arabic、5%的bovine serum albumin、及acetonitrile的使用為酵素反應的最佳模式,三者息息相關,未來測定LPL活性時應審慎評估。此分析方法的回收率介於108.73% - 114.36%之間,同日與異日間的精密度試驗,其相對標準偏差值分別在1.28%和2.91%之內,而偵測極限為4.53 nM。 本論文所建立的HPLC螢光偵測分析法,可成功應用於正常與糖尿病老鼠血漿中LPL活性之分析,我們進而發現糖尿病老鼠之血漿LPL活性和正常老鼠相比顯著降低了52.3%。未來希望此法能應用於不同生理與病理狀態下LPL活性的測定,進一步探討LPL活性和糖尿病病程及併發症的相關機制。
英文摘要 Lipoprotein lipase (LPL) is associated with the luminal side of capillaries and arteries where it hydrolyzes triglycerides in circulating lipoproteins to produce free fatty acids. It was valuable to determine LPL activity which was shown to vary in diseases and metabolic disorders. This research aim to develop a highly sensitive HPLC method using the fluorescent reagent, 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ), for derivatization of oleic acid (OA) liberated after triolein being hydrolyzed by LPL without sample extraction. The derivatized fatty acids could be adequately separated from interfering peaks. The substrate for LPL was loaded in with a saturated concentration of 10 mM, and bovine serum albumin (BSA) was found to be a key factor in LPL reactions. Gum Arabic (GA) was chosen for the emulsifier, but the used concentration as 1% was critical. Optimum condition for measuring the LPL activity was found when the produced OA dissolved in acetonitrile (MeCN). The accuracy values for the determination of LPL activity in 10 μL of rat post heparin plasma were 108.73 - 114.36%, and the intra- and inter-day precision values were within 1.28% and 2.91%, respectively. The limit of detection was about 4.53 nM. The proposed HPLC method was successfully applied to determine LPL activity in the post heparin plasma samples of normal and streptozotocin-induced diabetic rats. The LPL activity of diabetic rat was reduced about 52.3% compared with control. The established assay system is envisioned to be useful for determining LPL activity in different physiological and pathological conditions to clarify the relationship between LPL activity and diabetes mellitus.
論文目次 目錄 I 附圖目錄 III 附表目錄 V 縮寫表 VI 中文摘要 VII 英文摘要 VIII 第一章 序論 1 第一節 脂蛋白脂?的背景介紹 1 第二節 與脂蛋白脂?相關的疾病 10 第三節 目前脂蛋白脂?活性的測定方法 12 第四節 研究動機與目的 16 第二章 實驗材料與方法 17 第一節 實驗動物及材料 17 一、實驗動物 17 二、試藥 17 三、儀器 18 第二節 實驗方法 20 第一部份 脂蛋白脂?活性測定法之建立 20 一、游離脂肪酸的螢光衍生化與HPLC條件之探討 20 (1)油酸衍生化條件之選擇 20 (2)應用於LPL活性測定之油酸衍生化反應 21 (3)高效能液相層析儀之分析條件 22 二、受質乳化和酵素反應條件的最佳化 23 (1)酵素動力學模式的建立 24 (2)乳化劑種類及濃度的選擇 24 (3)白蛋白濃度與輔?的影響 24 (4)去蛋白溶媒的種類 25 三、分析方法之確效試驗 25 (1)檢量線線性(Linearity) 25 (2)準確度試驗(Accuracy) 26 (3)精密度試驗(Precision) 26 第二部分 分析方法的應用 27 一、實驗動物糖尿病的誘導 27 二、活體酵素來源的製備 28 三、活體脂蛋白脂?活性的分析 29 第三章 實驗結果與討論 30 第一部份 脂蛋白脂?活性活性測定法之建立 30 一、游離脂肪酸的螢光衍生化與HPLC條件之建立 30 (1)衍生化條件之選擇 31 (2)油酸之衍生化反應 33 (3)高效能液相層析儀之分析條件 33 二、受質乳化和酵素反應條件的最佳化 36 (1)酵素動力學模式的建立 36 (2)乳化劑種類及濃度的選擇 39 (3)白蛋白濃度與輔?的影響 51 (4)去蛋白溶媒的種類 57 三、分析方法之確效試驗 59 (1)檢量線線性(Linearity) 59 (2)準確度試驗(Accuracy) 63 (3)精密度試驗(Precision) 63 第二部分 分析方法的應用 64 一、生物檢體中脂蛋白脂?活性之測定 64 二、脂蛋白脂?活性與大鼠週齡之相關性 66 三、糖尿病大鼠脂蛋白脂?活性之測定 68 第四章 結論與未來展望 72 參考文獻 74
參考文獻 [1] L. A. Lehninger, L. D. Nelson, and M. M. Cox, Principles of Biochemistry, Worth, New York, 1993. [2] M. Hamosh and P. Hamosh, Molecular Aspects of Medicine 6 (1983) 199. [3] A. Bensadoun, Annual Review of Nutrition 11 (1991) 217. [4] P. F. Hahn, Science 98 (1943) 19. [5] C. B. Anfinsen, E. Boyle, and R. K. Brown, Science 115 (1952) 583. [6] D. S. Robinson and J. E. French, The Quarterly journal of experimental psychology 38 (1953) 233. [7] E. D. Korn, The Journal of biological chemistry 215 (1955) 15. [8] R. J. Havel and R. S. J. Gordon, The Journal of clinical investigation 39 (1960) 1777. [9] S. Santamarina-Fojo and K. A. Dugi, Current Opinion in Lipidology 5 (1994) 117. [10] I. J. Goldberg, Journal of Lipid Research 37 (1996) 693. [11] J. E. Braun and D. L. Severson, Biochemical Journal 287 (1992) 337. [12] C. F. Semenkovich, S. H. Chen, M. Wims, C. C. Luo, W. H. Li, and L. Chan, Journal of Lipid Research 30 (1989) 423. [13] R. Zechner, Current Opinion in Lipidology 8 (1997) 77. [14] J. D. Medh, S. L. Bowen, G. L. Fry, S. Ruben, M. Andracki, I. Inoue, J. M. Lalouel, D. K. Strickland, and D. A. Chappell, Journal of Biological Chemistry 271 (1996) 17073. [15] M. Merkel, Y. Kako, H. Radner, I. S. Cho, R. Ramasamy, J. D. Brunzell, I. J. Goldberg, and J. L. Breslow, Proceedings of the National Academy of Sciences of the United States of America 95 (1998) 13841. [16] U. Beisiegel, W. Weber, and G. Bengtsson-Olivecrona, Proceedings of the National Academy of Sciences of the United States of America 88 (1991) 8342. [17] S. Eisenberg, E. Sehayek, T. Olivecrona, and I. Vlodavsky, Journal of Clinical Investigation 90 (1992) 2013. [18] A. Nykjaer, M. Nielsen, A. Lookene, N. Meyer, H. Roigaard, M. Etzerodt, U. Beisiegel, G. Olivecrona, and J. Gliemann, Journal of Biological Chemistry 269 (1994) 31747. [19] A. M. van Bennekum, Y. Kako, P. H. Weinstock, E. H. Harrison, R. J. Deckelbaum, I. J. Goldberg, and W. S. Blaner, Journal of Lipid Research 40 (1999) 565. [20] W. Sattler, S. Levak-Frank, H. Radner, G. M. Kostner, and R. Zechner, Biochemical Journal 318 (1996) 15. [21] P. H. Weinstock, C. L. Bisgaier, K. Aalto-Setala, H. Radner, R. Ramakrishnan, S. Levak-Frank, A. D. Essenburg, R. Zechner, and J. L. Breslow, Journal of Clinical Investigation 96 (1995) 2555. [22] G. Wu, in Department of Medical Biosciences, Physiological Chemistry, Vol. Ph. D, Ume? University, Ume?, Sweden, 2004. [23] G. Wu, G. Olivecrona, and T. Olivecrona, Journal of Biological Chemistry 278 (2003) 11925. [24] T. Ruge, G. Wu, T. Olivecrona, and G. Olivecrona, International Journal of Biochemistry & Cell Biology 36 (2004) 320. [25] A. Cryer, International Journal of Biochemistry 13 (1981) 525. [26] B. K. Speake, S. M. Parkin, and D. S. Robinson, Biochemical Society Transactions 13 (1985) 29. [27] K. Preiss-Landl, R. Zimmermann, G. Hammerle, and R. Zechner, Current Opinion in Lipidology 13 (2002) 471. [28] M. Berg?, G. Wu, T. Ruge, and T. Olivecrona, Journal of Biological Chemistry 277 (2002) 11927. [29] G. Wu, P. Brouckaert, and T. Olivecrona, American Journal of Physiology - Endocrinology & Metabolism 286 (2004) E711. [30] R. L. Patten, Journal of Biological Chemistry 245 (1970) 5577. [31] G. Friedman, T. Chajek-Shaul, O. Stein, L. Noe, J. Etienne, and Y. Stein, Biochimica et Biophysica Acta 877 (1986) 112. [32] D. L. Severson, R. Carroll, A. Kryski, Jr., and I. Ramirez, Biochemical Journal 248 (1987) 289. [33] R. H. Eckel, New England Journal of Medicine 320 (1989) 1060. [34] H. Wong, D. Yang, J. S. Hill, R. C. Davis, J. Nikazy, and M. C. Schotz, Proceedings of the National Academy of Sciences of the United States of America 94 (1997) 5594. [35] T. G. Kirchgessner, K. L. Svenson, A. J. Lusis, and M. C. Schotz, Journal of Biological Chemistry 262 (1987) 8463. [36] K. L. Wion, T. G. Kirchgessner, A. J. Lusis, M. C. Schotz, and R. M. Lawn, Science 235 (1987) 1638. [37] M. Senda, K. Oka, W. V. Brown, P. K. Qasba, and Y. Furuichi, Proceedings of the National Academy of Sciences of the United States of America 84 (1987) 4369. [38] Z. S. Derewenda and C. Cambillau, Journal of Biological Chemistry 266 (1991) 23112. [39] R. C. Davis, H. Wong, J. Nikazy, K. Wang, Q. Han, and M. C. Schotz, Journal of Biological Chemistry 267 (1992) 21499. [40] H. Wong, R. C. Davis, T. Thuren, J. W. Goers, J. Nikazy, M. Waite, and M. C. Schotz, Journal of Biological Chemistry 269 (1994) 10319. [41] M. S. Nielsen, J. Brejning, R. Garcia, H. Zhang, M. R. Hayden, S. Vilaro, and J. Gliemann, Journal of Biological Chemistry 272 (1997) 5821. [42] T. L. McIlhargey, Y. Yang, H. Wong, and J. S. Hill, Journal of Biological Chemistry 278 (2003) 23027. [43] A. J. Scheen, Revue Medicale de Liege 54 (1999) 87. [44] J. R. Mead, S. A. Irvine, and D. P. Ramji, Journal of Molecular Medicine 80 (2002) 753. [45] L. M. Keilson, C. P. Vary, D. L. Sprecher, and R. Renfrew, Annals of Internal Medicine 124 (1996) 425. [46] L. Baum, H. Wiebusch, and C. P. Pang, Microscopy Research & Technique 50 (2000) 291. [47] M. Merkel, R. H. Eckel, and I. J. Goldberg, Journal of Lipid Research 43 (2002) 1997. [48] J. Schneider, A. Liesenfeld, R. Mordasini, R. Schubotz, P. Zofel, F. Kubel, C. Vandre-Plozzitzka, and H. Kaffarnik, Atherosclerosis 57 (1985) 281. [49] E. A. Nikkil?, M. R. Taskinen, and J. K. Huttunen, Hormone & Metabolic Research 10 (1978) 220. [50] P. O'Looney, M. Vander Maten, and G. V. Vahouny, Journal of Biological Chemistry 258 (1983) 12994. [51] P. O'Looney, D. Irwin, P. Briscoe, and G. V. Vahouny, Journal of Biological Chemistry 260 (1985) 428. [52] J. D. Brunzell, D. Porte, Jr., and E. L. Bierman, Metabolism: Clinical & Experimental 24 (1975) 1123. [53] P. J. Randle, P. B. Garland, C. N. Hales, and E. A. Newsholme, Lancet (1963) 785. [54] J. K. Kim, J. K. Wi, and J. H. Youn, Diabetes 45 (1996) 651. [55] S. Levak-Frank, H. Radner, A. Walsh, R. Stollberger, G. Knipping, G. Hoefler, W. Sattler, P. H. Weinstock, J. L. Breslow, and R. Zechner, Journal of Clinical Investigation 96 (1995) 976. [56] D. R. Jensen, I. R. Schlaepfer, C. L. Morin, D. S. Pennington, T. Marcell, S. M. Ammon, A. Gutierrez-Hartmann, and R. H. Eckel, American Journal of Physiology 273 (1997) R683. [57] J. K. Kim, J. J. Fillmore, Y. Chen, C. Yu, I. K. Moore, M. Pypaert, E. P. Lutz, Y. Kako, W. Velez-Carrasco, I. J. Goldberg, J. L. Breslow, and G. I. Shulman, Proceedings of the National Academy of Sciences of the United States of America 98 (2001) 7522. [58] L. K. Pulawa and R. H. Eckel, Current Opinion in Clinical Nutrition & Metabolic Care 5 (2002) 569. [59] L. Liu and D. L. Severson, Canadian Journal of Physiology & Pharmacology 72 (1994) 1259. [60] H. Lithell, J. Boberg, K. Hellsing, S. Ljunghall, G. Lundqvist, B. Vessby, and L. Wide, European Journal of Clinical Investigation 11 (1981) 3. [61] K. Kirkeby, Acta Endocrinologica 59 (1968) 555. [62] P. Hansson, G. Nordin, and P. Nilsson-Ehle, Biochimica et Biophysica Acta 753 (1983) 364. [63] W. Lutz, Acta Medica Polona 20 (1979) 131. [64] L. Verschoor, R. G. Baggen, H. Jansen, and J. C. Birkenhager, Journal of Clinical Endocrinology & Metabolism 56 (1983) 592. [65] M. K. Chan, J. W. Persaud, Z. Varghese, and J. F. Moorhead, Australian & New Zealand Journal of Medicine 14 (1984) 841. [66] E. Levy, E. Ziv, H. Bar-On, and E. Shafrir, Biochimica et Biophysica Acta 1043 (1990) 259. [67] R. W. Mahley, K. H. Weisgraber, T. L. Innerarity, and S. C. Rall, Jr., JAMA 265 (1991) 78. [68] O. J. Pykalisto, P. H. Smith, and J. D. Brunzell, Journal of Clinical Investigation 56 (1975) 1108. [69] E. A. Nikkil?, J. K. Huttunen, and C. Ehnholm, Diabetes 26 (1977) 11. [70] M. R. Taskinen, E. A. Nikkil?, T. Kuusi, and K. Harmo, Diabetologia 22 (1982) 46. [71] J. Jeppesen, C. B. Hollenbeck, M. Y. Zhou, A. M. Coulston, C. Jones, Y. D. Chen, and G. M. Reaven, Arteriosclerosis, Thrombosis & Vascular Biology 15 (1995) 320. [72] P. Maheux, S. Azhar, P. A. Kern, Y. D. Chen, and G. M. Reuven, Diabetologia 40 (1997) 850. [73] M. R. Sartippour and G. Renier, Diabetes 49 (2000) 597. [74] H. Yamazaki, M. Arai, S. Matsumura, K. Inoue, and T. Fushiki, American Journal of Physiology - Endocrinology & Metabolism 283 (2002) E536. [75] C. S. Wang, A. Kuksis, and F. Manganaro, Lipids 17 (1982) 278. [76] J. E. Bauer, Artery 15 (1988) 272. [77] R. Gupta, P. Rathi, N. Gupta, and S. Bradoo, Biotechnology & Applied Biochemistry 37 (2003) 63. [78] F. Beisson, A. Tiss, C. Rivi?re, and R. Verger, European Journal of Lipid Science and Technology 102 (2000) 133. [79] A. Hoppe and R. R. Theimer, Phytochemistry 42 (1996) 973. [80] P. Nilsson-Ehle and M. C. Schotz, Journal of Lipid Research 17 (1976) 536. [81] M. Duque, M. Graupner, H. Stutz, I. Wicher, R. Zechner, F. Paltauf, and A. Hermetter, Journal of Lipid Research 37 (1996) 868. [82] P. Belfrage and M. Vaughan, Journal of Lipid Research 10 (1969) 341. [83] S. W. Ball, J. R. Bailey, J. M. Stewart, C. M. Vogels, and S. A. Westcott, Canadian Journal of Physiology & Pharmacology 80 (2002) 205. [84] Y. Eguchi, Biomedical Chromatography 16 (2002) 500. [85] D. A. Nickerson, S. L. Taylor, K. M. Weiss, A. G. Clark, R. G. Hutchinson, J. Stengard, V. Salomaa, E. Vartiainen, E. Boerwinkle, and C. F. Sing, Nature Genetics 19 (1998) 233. [86] J. Hoh and S. E. Hodge, Human Heredity 50 (2000) 359. [87] A. R. Templeton, K. M. Weiss, D. A. Nickerson, E. Boerwinkle, and C. F. Sing, Genetics 156 (2000) 1259. [88] M. L. Baginsky and W. V. Brown, Journal of Lipid Research 18 (1977) 423. [89] M. L. Baginsky and W. V. Brown, Journal of Lipid Research 20 (1979) 548. [90] A. Sparreboom, O. van Tellingen, M. T. Huizing, W. J. Nooijen, and J. H. Beijnen, Journal of Chromatography B: Biomedical Applications 681 (1996) 355. [91] Y. C. Tsai, T. H. Liao, and J. A. Lee, Analytical Biochemistry 319 (2003) 34. [92] D. M. Foster and M. Berman, Journal of Lipid Research 22 (1981) 506. [93] R. A. Burdette and D. M. Quinn, Journal of Biological Chemistry 261 (1986) 12016. [94] B. Surinenaite, V. Bendikiene, B. Juodka, I. Bachmatova, and L. Marcinkevichiene, Biotechnology & Applied Biochemistry 36 (2002) 47. [95] R. M. Archibald, Journal of Biological Chemistry 165 (1946) 443. [96] R. P. Yadav, R. K. Saxena, R. Gupta, and W. S. Davidson, Biotechnology & Applied Biochemistry 28 (1998) 243. [97] J. C. LaRosa, R. I. Levy, P. Herbert, S. E. Lux, and D. S. Fredrickson, Biochemical & Biophysical Research Communications 41 (1970) 57. [98] T. Tsujita, Y. Matsuura, and H. Okuda, Journal of Lipid Research 37 (1996) 1481. [99] E. J. Blanchette-Mackie and R. O. Scow, Journal of Lipid Research 17 (1976) 57. [100] F. Karpe, T. Olivecrona, G. Walldius, and A. Hamsten, Journal of Lipid Research 33 (1992) 975. [101] L. I. Lobo and D. C. Wilton, Biochemical Journal 321 (1997) 829. [102] D. M. Bier and R. J. Havel, Journal of Lipid Research 11 (1970) 565. [103] R. Sharma, Y. Chisti, and U. C. Banerjee, Biotechnology Advances 19 (2001) 627. [104] S. Schneider, U. Schramm, A. Schreyer, H. P. Buscher, W. Gerok, and G. Kurz, Journal of Lipid Research 32 (1991) 1755. [105] J. Kovar, V. Fejfarova, T. Pelikanova, and R. Poledne, Physiological Research 53 (2004) 61. [106] B. Rodrigues, M. C. Cam, J. Kong, R. K. Goyal, and J. H. McNeill, Cardiovascular Research 34 (1997) 199.

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系統識別號 U0007-1704200714541729
論文名稱(中文) 織蛋白去乙醯化酶抑制劑(丙基戊酸)對急性白血病細胞生長、凋亡和分化特性的研究
論文名稱(英文) Characterization of the Histone Deacetylase Inhibitor (Valproic Acid) on the Growth, Apoptosis and Differentiation of Acute Myeloid Leukemia
校院名稱 臺北醫學大學
系所名稱(中) 醫學檢驗生物技術學研究所
系所名稱(英) Graduate Institute of Biomedical Technology
學年度 93
學期 2
出版年 94
研究生(中文) 林秀盆
學號 G160091010
學位類別 碩士
語文別 中文
口試日期
論文頁數 60頁
口試委員 指導教授-劉興璟 博士
指導教授-林建煌 教授
關鍵字(中) 織蛋白去乙醯化酶
抑制劑
丙基戊酸
凋亡作用
分化作用
死亡接受器
JNK 路徑
關鍵字(英) Epigenetic
histone deacetylase inhibitors
valproic acid
differentiation
death receptor
JNK pathway
apoptosis
學科別分類
中文摘要 論文摘要 論文名稱:織蛋白去乙醯化酶抑制劑(丙基戊酸)對急性白血病細胞生長、凋亡和分化特性的研究 研究所名稱:台北醫學大學生物醫學技術研究所 研究生姓名:林秀盆 畢業時間:93學年度第1學期 指導教授:劉興璟 助理教授 醫學研究所 林建煌 教授 醫學研究所 Epigenetic調控基因表現,對造血作用與白血病的形成有密切關聯。組織蛋白去乙醯化酶是epigenetic 調控的重要成員之一。本研究在探討組織蛋白去乙醯化酶與白血病的增生、細胞分化、細胞凋亡的關係。發現組織蛋白去乙醯化酶抑制劑 (酪酸鈉、酪酸苯丙基戊酸和SAHA),能有效的抑制人類急性骨髓性白血病細胞株的增生,誘導細胞分化或是促進細胞凋亡作用,酪酸鈉、酪酸苯和SAHA使細胞週期停滯G0/G1期,而丙基戊酸不影響細胞週期。同時發現組織蛋白去乙醯化酶抑制劑,能誘導人類急性骨髓性白血病細胞株走向單核球分化,酪酸鈉能有效誘導CD11c表現,丙基戊酸對誘導CD13表現最好,而四種藥劑都能有效誘導CD14表現,並增加α-Naphthyl Acetate Esterase (NAE) 陽性細胞,但NBT (nitroblue tetrazolium) 還原反應細胞很微量。因此我們針對丙基戊酸,治療人類急性骨髓性白血病單核球細胞株 (THP-1) 的影響,研究發現經由細胞週期蛋白質表現,丙基戊酸誘導 cyclin D1、P21和P27表現, cyclin D1 和 P21誘導骨髓性細胞走向分化。而細胞走向分化可能跟核酸c-Jun和蛋白質表現有關,及 Bcl-2、bid、caspase-9 和 caspase-3 蛋白質表現,誘導骨髓性細胞凋亡作用。凋亡路徑可能是啟動死亡接受器 (death receptor) 與粒線體路徑,而經由 tBid 使兩者路徑有交互聯結,而丙基戊酸活化 JNK 路徑對誘導骨髓性細胞分化作用,可能是重要的關鍵。
英文摘要 Abstract Epigenetic control of gene expression plays an important role in hematopoiesis and leukemogenesis. One of the major components in epigenetic regulation of gene expression is histone acetylation.Recent studies have shown that histone deacetylase inhibitors (HDACIs) might be useful for treating hematopoietic malignancies. However the effects of these HDACIs on human acute myeloid leukemia (AML) have not been studied comprehensively. In this study, we examined the effects of several clinically available HDACIs on the proliferation, differentiation and apoptosis of AML in vitro. First HL-60 cells were treated with increasing concentrations of sodium butyrate (SB), a prototypic HDACI, phenylbutyrate (PB), and suberoylanilide hydroxamic acid (SAHA), an HDACI in phase-I clinical trials, valproic acid (VA) , a clinically available agent for neurological disorders.We found that SB, PB, and SAHA were able to arrest cell cycle at G0/G1 phase, but VA did not. SB and VA significantly induced the expression of CD11c and CD13, respectively. CD14 expression was upregulated by all four agents, All four agents induced mild neutrophilic and marked monocytic differentiation evidenced by nitroblue tetrazolium (NBT) tests andα-Naphthyl Acetate Esterase (NAE) staining,respectively. Further elucidation of VA induced apoptosis through both mitochondrial and death receptor pathway.VA induced cyclin D1, p21 and p27 expression, cycle D1 and p21 might relate cell differentiation,so cell cycle did not arrest at G0/G1 phase in 48H.Valproic acid activated JNK pathway might play an important role in monocytic differentiation.
論文目次 目錄 目錄 i 論文摘要 v Abstract vii 縮寫表 viii 第壹章、 緒論 1 ㄧ、白血病 (leukemia) 1 二、急性骨髓性白血病 (Acute myeloid leukemia) 1 三、Epigenetics 改變與腫瘤的形成 2 (1) Epigenetics 2 (2) DNA 甲基轉位酶 DNA methyltransferases (DNMTs) 3 (3) 組織蛋白去乙醯化酶 (histone deacetylase) 4 第貳章 材料與方法 10 第一節、藥劑 10 第二節、細胞培養 (Cell culture) 10 第三節、細胞計數 11 一、 Trypan blue exclusion 11 二、MTT (3-[4,5-Dimethyl-2-thiazolyl]-2,5-diphenyltetrazolium bromide) 11 第四節、細胞週期 (cell cycle) 之分析 11 第五節、影響細胞株增殖 12 第六節、細胞形態 (Cell morphology) 12 第七節、特殊細胞化學染色 12 一、Nitroblue tetrazolium (NBT) test 13 二、α-NAE stain 13 第八節、細胞表面標誌分析 (Cell surface marker analysis) 13 第九節、測細胞凋亡 (detect apoptosis) 14 第十節、粒腺體通透膜電位差分析 14 第十一節、 基因表現 (Gene expression) 14 ㄧ、Total RNA extraction 14 二、RT-PCR (reverse transcriptase-polymerase chain reaction) 15 三、SYBR Green染色 16 四、Real-time PCR 方法 16 第十二節、細胞核及細胞質蛋白質的萃取 16 一、蛋白質萃取 16 二、以 Bradford method 定量蛋白質濃度 17 三、西方墨點法 (硫酸十二酯鈉-聚丙烯醯胺凝膠 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) 17 第參章 實驗結果與分析 19 第一節、組織蛋白去乙醯化酶抑制劑影響 HL-60 細胞株增殖 19 第二節、組織蛋白去乙醯化酶抑制劑對細胞週期的影響 20 第三節、誘導細胞凋亡及可能凋亡路線 21 第四節、組織蛋白去乙醯化酶抑制劑誘導細胞分化 22 一、細胞形態的改變 (Cell morphological change) 22 二、α-Naphthyl Acetate Esterase(NAE) 染色 22 第五節、丙基戊酸對基因及相關蛋白質表現的影響 23 第肆章、結果討論 24 References 27 附表 32 Table 1. FAB classification of acute myeloid leukemias 32 Table 2. WHO classification of acute myeloid leukemias 33 Table 3. Oligonucleotide primers used for this study 34 Table 4. Effects of VA on the cell cycle 35 Table 5. Effects of HDACIs on the percentage of NBT reducing-and NAE stain. 36 附圖 37 Fig. 1. Effect of various HDACIs on the viability in HL-60 cells. 37 Fig. 2. Antiproliferation in HL-60 cells by HDACs. 38 Fig. 3. Effect cell cycle by HDACIs in HL-60 cells. 39 Fig. 4 Valproic acid does not arrest cell cycle. 40 Fig. 5 Response of VA-induced cell cycle-related proteins in THP-1 cells. 41 Fig. 6 Apoptosis and related pathway assay. 42 Fig. 6 (C) Reduction of mitochondrial transmembrane potential (△Ψm) correlates in a dose dependent manner. 43 Fig. 7 (A) Effect of Bcl-2 family expression by VA. 44 Fig. 7 (B) Time-dependent expression of caspase. 45 Fig. 8 (A) Morphological changes by HDACIs. 46 Fig. 8 (B) Induced Monocytic Differentiation of THP-1 and HL-60 Cells by VA. 47 Fig. 8 (C) Effects of HDACIs on the percentage of α-Naphthyl Acetate Esterase (NAE) stain on. HL-60 cells. 48 Fig. 9 Effects of HDACIs on the expression of CD11b, CD11c CD13 and CD14 on HL-60 cells. 49 Fig. 10 (A) Time course—response of VA-regulation in THP-1 cells for gene expression. 50 Fig. 10 (B) Time course—response of VA-regulation in THP-1 cells for MAPK protein expression. 51 Fig. 11 Proposed mechanism for monocytic differentiation by JNK Pathway. 52
參考文獻 References Archer, S.Y., J.J. Johnson, H.J. Kim, and R.A. Hodin. 2001. p21 gene regulation during enterocyte differentiation. J Surg Res. 98:4-8. Arellano, M., and S. Moreno. 1997. Regulation of CDK/cyclin complexes during the cell cycle. Int J Biochem Cell Biol. 29:559-73. Aronis, A., J.A. Melendez, O. Golan, S. Shilo, N. Dicter, and O. Tirosh. 2003. Potentiation of Fas-mediated apoptosis by attenuated production of mitochondria-derived reactive oxygen species. Cell Death Differ. 10:335-44. Bacon, C.L., H.C. Gallagher, J.C. Haughey, and C.M. Regan. 2002. Antiproliferative action of valproate is associated with aberrant expression and nuclear translocation of cyclin D3 during the C6 glioma G1 phase. J Neurochem. 83:12-9. Bartkova, J., E. Rajpert-de Meyts, N.E. Skakkebaek, and J. Bartek. 1999. D-type cyclins in adult human testis and testicular cancer: relation to cell type, proliferation, differentiation, and malignancy. J Pathol. 187:573-81. Bird, A. 2002. DNA methylation patterns and epigenetic memory. Genes Dev. 16:6-21. Bojic, U., K. Ehlers, U. Ellerbeck, C.L. Bacon, E. O''Driscoll, C. O''Connell, V. Berezin, A. Kawa, E. Lepekhin, E. Bock, C.M. Regan, and H. Nau. 1998. Studies on the teratogen pharmacophore of valproic acid analogues: evidence of interactions at a hydrophobic centre. Eur J Pharmacol. 354:289-99. Chen, G., P. Yuan, D.B. Hawver, W.Z. Potter, and H.K. Manji. 1997. Increase in AP-1 transcription factor DNA binding activity by valproic acid. Neuropsychopharmacology. 16:238-45. Chen, Y., R.P. Sharma, R.H. Costa, E. Costa, and D.R. Grayson. 2002. On the epigenetic regulation of the human reelin promoter. Nucleic Acids Res. 30:2930-9. Chopin, V., R.A. Toillon, N. Jouy, and X. Le Bourhis. 2004. P21(WAF1/CIP1) is dispensable for G1 arrest, but indispensable for apoptosis induced by sodium butyrate in MCF-7 breast cancer cells. Oncogene. 23:21-9. Csordas, A. 1990. On the biological role of histone acetylation. Biochem J. 265:23-38. Dumont, C., A. Durrbach, N. Bidere, M. Rouleau, G. Kroemer, G. Bernard, F. Hirsch, B. Charpentier, S.A. Susin, and A. Senik. 2000. Caspase-independent commitment phase to apoptosis in activated blood T lymphocytes: reversibility at low apoptotic insult. Blood. 96:1030-8. Garber, K. 2002. Breaking the silence: the rise of epigenetic therapy. J Natl Cancer Inst. 94:874-5. Gong, J., F. Traganos, and Z. Darzynkiewicz. 1995. Growth imbalance and altered expression of cyclins B1, A, E, and D3 in MOLT-4 cells synchronized in the cell cycle by inhibitors of DNA replication. Cell Growth Differ. 6:1485-93. Gottlicher, M., S. Minucci, P. Zhu, O.H. Kramer, A. Schimpf, S. Giavara, J.P. Sleeman, F. Lo Coco, C. Nervi, P.G. Pelicci, and T. Heinzel. 2001. Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells. Embo J. 20:6969-78. Herman, J. 1999. Hypermethylation of tumor suppressor genes in cancer. Seminars in Cancer Biology. 9:359-67,. Hoessly, M.C., R.M. Rossi, and S.A. Fischkoff. 1989. Factors responsible for variable reported lineages of HL-60 cells induced to mature with butyric acid. Cancer Res. 49:3594-7. Hong, J., K. Ishihara, K. Yamaki, K. Hiraizumi, T. Ohno, J.W. Ahn, O. Zee, and K. Ohuchi. 2003. Apicidin, a histone deacetylase inhibitor, induces differentiation of HL-60 cells. Cancer Lett. 189:197-206. Insinga, A., S. Monestiroli, S. Ronzoni, V. Gelmetti, F. Marchesi, A. Viale, L. Altucci, C. Nervi, S. Minucci, and P.G. Pelicci. 2005. Inhibitors of histone deacetylases induce tumor-selective apoptosis through activation of the death receptor pathway. Nat Med. 11:71-6. Ishizaki, Y., M.D. Jacobson, and M.C. Raff. 1998. A role for caspases in lens fiber differentiation. J Cell Biol. 140:153-8. Jaboin, J., J. Wild, H. Hamidi, C. Khanna, C.J. Kim, R. Robey, S.E. Bates, and C.J. Thiele. 2002. MS-27-275, an inhibitor of histone deacetylase, has marked in vitro and in vivo antitumor activity against pediatric solid tumors. Cancer Res. 62:6108-15. Jaenisch, R., and A. Bird. 2003. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet. 33 Suppl:245-54. Johnstone, R.W. 2002. Histone-deacetylase inhibitors: novel drugs for the treatment of cancer. Nat Rev Drug Discov. 1:287-99. Jones, P.A., and S.B. Baylin. 2002. The fundamental role of epigenetic events in cancer. Nat Rev Genet. 3:415-28. Jones, P.A., and P.W. Laird. 1999. Cancer epigenetics comes of age. Nat Genet. 21:163-7. Kato, J.Y., and C.J. Sherr. 1993. Inhibition of granulocyte differentiation by G1 cyclins D2 and D3 but not D1. Proc Natl Acad Sci U S A. 90:11513-7. Kawagoe, R., H. Kawagoe, and K. Sano. 2002. Valproic acid induces apoptosis in human leukemia cells by stimulating both caspase-dependent and -independent apoptotic signaling pathways. Leuk Res. 26:495-502. Kiess, M., R.M. Gill, and P.A. Hamel. 1995. Expression of the positive regulator of cell cycle progression, cyclin D3, is induced during differentiation of myoblasts into quiescent myotubes. Oncogene. 10:159-66. Kitamura, K., S. Hoshi, M. Koike, H. Kiyoi, H. Saito, and T. Naoe. 2000. Histone deacetylase inhibitor but not arsenic trioxide differentiates acute promyelocytic leukaemia cells with t(11;17) in combination with all-trans retinoic acid. Br J Haematol. 108:696-702. Li, C.R., W.L. Liu, M. Huang, J.N. Deng, H.Y. Sun, and J.F. Zhou. 2004. [Effect of sodium butyrate in combination with ATRA on the proliferation/differentiation of MDS cell line SKM-1.]. Zhongguo Shi Yan Xue Ye Xue Za Zhi. 12:601-5. Louis, M., R.R. Rosato, L. Brault, S. Osbild, E. Battaglia, X.H. Yang, S. Grant, and D. Bagrel. 2004. The histone deacetylase inhibitor sodium butyrate induces breast cancer cell apoptosis through diverse cytotoxic actions including glutathione depletion and oxidative stress. Int J Oncol. 25:1701-11. Marks, P.A. 2004. The mechanism of the anti-tumor activity of the histone deacetylase inhibitor, suberoylanilide hydroxamic acid ( SAHA ). Cell Cycle. 3:534-5. Marks, P.A., V.M. Richon, T. Miller, and W.K. Kelly. 2004. Histone deacetylase inhibitors. Adv Cancer Res. 91:137-68. Melki, J.R., P.C. Vincent, and S.J. Clark. 1999. Concurrent DNA hypermethylation of multiple genes in acute myeloid leukemia. Cancer Res. 59:3730-40. Melnick, A., and J.D. Licht. 2002. Histone deacetylases as therapeutic targets in hematologic malignancies. Curr Opin Hematol. 9:322-32. Mitsiades, C.S., V. Poulaki, and N. Mitsiades. 2003. The role of apoptosis-inducing receptors of the tumor necrosis factor family in thyroid cancer. J Endocrinol. 178:205-16. Okado, N. 1999. [Mechanisms for formation and maintenance of synapses mediated by biogenic amines: pathogenesis and therapy of mental retardation and developmental disabilities by genetic and epigenetic factors]. Kaibogaku Zasshi. 74:351-62. Pazin, M.J., and J.T. Kadonaga. 1997. What''s up and down with histone deacetylation and transcription? Cell. 89:325-8. Rezaei, A., M. Adib, F. Mokarian, M. Tebianian, and R. Nassiri. 2003. Leukemia markers expression of peripheral blood vs bone marrow blasts using flow cytometry. Med Sci Monit. 9:CR359-62. Richardson, B., and R. Yung. 1999. Role of DNA methylation in the regulation of cell function. J Lab Clin Med. 134:333-40. Ruefli, A.A., M.J. Ausserlechner, D. Bernhard, V.R. Sutton, K.M. Tainton, R. Kofler, M.J. Smyth, and R.W. Johnstone. 2001. The histone deacetylase inhibitor and chemotherapeutic agent suberoylanilide hydroxamic acid ( SAHA ) induces a cell-death pathway characterized by cleavage of Bid and production of reactive oxygen species. Proc Natl Acad Sci U S A. 98:10833-8. Ruefli, A.A., M.J. Smyth, and R.W. Johnstone. 2000. HMBA induces activation of a caspase-independent cell death pathway to overcome P-glycoprotein-mediated multidrug resistance. Blood. 95:2378-85. Singal, R., and G.D. Ginder. 1999. DNA methylation. Blood. 93:4059-70. Sordet, O., C. Rebe, S. Plenchette, Y. Zermati, O. Hermine, W. Vainchenker, C. Garrido, E. Solary, and L. Dubrez-Daloz. 2002. Specific involvement of caspases in the differentiation of monocytes into macrophages. Blood. 100:4446-53. Tang, R., A.M. Faussat, P. Majdak, J.Y. Perrot, D. Chaoui, O. Legrand, and J.P. Marie. 2004. Valproic acid inhibits proliferation and induces apoptosis in acute myeloid leukemia cells expressing P-gp and MRP1. Leukemia. 18:1246-51. Taylor, S.M., and P.A. Jones. 1982. Mechanism of action of eukaryotic DNA methyltransferase. Use of 5-azacytosine-containing DNA . J Mol Biol. 162:679-92. Tittle, T.V., B.A. Schaumann, J.E. Rainey, and K. Taylor. 1992. Segregation of the growth slowing effects of valproic acid from phenytoin and carbamazepine on lymphoid tumor cells. Life Sci. 50:PL79-83. Toyota, M., F. Itoh, and K. Imai. 2000. DNA methylation and gastrointestinal malignancies: functional consequences and clinical implications. J Gastroenterol. 35:727-34. Toyota, M., K.J. Kopecky, M.O. Toyota, K.W. Jair, C.L. Willman, and J.P. Issa. 2001. Methylation profiling in acute myeloid leukemia. Blood. 97:2823-9. Vardiman, J.W., N.L. Harris, and R.D. Brunning. 2002. The World Health Organization (WHO) classification of the myeloid neoplasms. Blood. 100:2292-302. Wang, Q., X. Wang, and G.P. Studzinski. 2003. Jun N-terminal kinase pathway enhances signaling of monocytic differentiation of human leukemia cells induced by 1,25-dihydroxyvitamin D3. J Cell Biochem. 89:1087-101. Zermati, Y., C. Garrido, S. Amsellem, S. Fishelson, D. Bouscary, F. Valensi, B. Varet, E. Solary, and O. Hermine. 2001. Caspase activation is required for terminal erythroid differentiation. J Exp Med. 193:247-54.

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系統識別號 U0007-1704200714542184
論文名稱(中文) 靈芝子實體纖維之幾丁質及幾丁聚醣之製備
論文名稱(英文) Preparation of Sacchachitin and Sacchachitosan from the Residue of Ganoderma Fruiting Bodies
校院名稱 臺北醫學大學
系所名稱(中) 口腔科學研究所
系所名稱(英) Graduate Institute of Oral Science
學年度 93
學期 2
出版年 94
研究生(中文) 車立雯
學號 M214092003
學位類別 碩士
語文別 中文
口試日期
論文頁數 99頁
口試委員 指導教授-陳建中
關鍵字(中) 靈芝
靈芝幾丁質
靈芝幾丁聚醣
去乙醯值
關鍵字(英) Ganoderma
Sacchachitin
Sacchachitosan
Degree of de-acetylation
學科別分類
中文摘要 靈芝為傳統常用之中藥,為真菌類重要成員,其形體可分為菌柄及子實體兩部分,靈芝子實體的成份除了高分子多醣體、三?類、腺?和小分子蛋白外,其餘大部分的纖維均為幾丁質,因此非常適合作為生產幾丁質及其衍生物之原料來源。目前工業上之幾丁質等衍生物來源,主要由蝦、蟹殼經酸鹼溶液多項步驟反應所製得,由於蝦蟹殼來源分佈廣泛,且因種類、大小、區域等差異,使得幾丁質等衍生物之物化性質不盡相同。 本研究分別先將子實體殘渣經超微粒研磨機處理微小化後,再針對靈芝子實體纖維之衍生物:靈芝幾丁質、靈芝幾丁聚醣進行萃取,並在不同條件下,以H2O2為變因進行脫色處理來決定製程之標準化。進而利用膠體滴定法、IR、NMR測得其靈芝幾丁質、幾丁聚醣之去乙醯值及結構分析,再以IV測其分子量;在型態學上則以SEM觀察其巨觀之狀態。而針對靈芝幾丁聚醣部分,則再進行A.A.抗菌性試驗。 本研究之目的在於期望能藉由此製程進而提升靈芝的用途至幾丁質、幾丁聚醣等具開發性潛力之成份,並提供其更加安全、穩定的來源及附加價值高的商業性利用。
英文摘要 As an important member of fungi, Ganoderma has two main parts: the stems and the fruiting bodies. Besides polysaccharide, triterpene, nucleoside, and protein, the main component of the Ganoderma fruiting bodies is chitin, hence an excellent sources for chitin and its derivatives. Presently, through multiple and complicated steps of alkali and acid reactions, chitin and its derivatives were produced with crustacean shells as major sources. As crustacean shells may have many variables, such as variety, size, and region, the physic-chemical properties of chitin and its derivatives obtained from these sources might be different. To the contrary, Ganoderma can be farmed and harvested in controlled environments as a much more reliable biomaterial sources. Optimized preparation procedures for chitin from Ganoderma were proposed. Beginning with the grinding of the Ganoderma fruiting bodies, the product powder was treated with sodium hydroxide followed by the separation and bleaching processes. Depending on the procedures, the final products are sacchachitin and sacchachitosan. Degree of de-acetylation, the chemical structure of saccharchitin and its derivatives, were analyzed using the PVSK titration method, IR, and NMR. The molecular weight was then determined using viscosity meter, and the morphology was investigated using SEM. An antibiotic test for sacchachitosan was undertaken with A. actinomycetemcomitans. The purpose of this study is to ensure the full utilizations of Ganoderma, to produce not only chitin but also other high valued products. Furthermore, we provided a much safer and steadier sources materials, through optimized procedures.
論文目次 目錄 致 謝 I 中文摘要 II ABSTRACT IV 目錄 VI 表目錄 X 圖目錄 XI 第一章 緒論 13 1.1 研究動機與其重要性 13 1.2 研究目的 15 1.3 研究假設 16 第二章 文獻回顧 17 2.1 靈芝 17 2.1.1 生長與栽培 17 2.1.2 松杉靈芝的形態 18 2.1.3 靈芝的生理活性成分 19 2.2 幾丁質(CHITIN)與幾丁聚醣(CHITOSAN) 22 2.2.1 幾丁質與幾丁聚醣的由來 22 2.2.2 幾丁質與幾丁聚醣的分布 23 2.2.3 幾丁質與幾丁聚醣的結構 23 2.2.4 幾丁質之種類 24 2.2.5 幾丁質之製備 25 2.2.6 幾丁聚醣之製備 26 2.2.7 幾丁質與幾丁聚醣之生合成 27 2.2.8 幾丁質與幾丁聚醣之溶解特性 27 2.2.9 幾丁質與幾丁聚醣之應用 28 2.3 過氧化氫(HYDROGEN PEROXIDE,H2O2)漂白機制 31 2.3.1 過氧化氫在鹼性環境中之漂白機制 31 2.3.2 殘留過氧化氫之檢查法 33 2.4 奈米微小化技術 34 2.4.1 何謂奈米 34 2.4.2 奈米材料的結構效應 35 2.4.3 奈米材料的表面效應 35 2.4.4 奈米材料的體積效應 35 第三章 研究材料與方法 37 3.1 材料與試劑 37 3.2 儀器設備 38 3.3 研究方法 39 3.3.1 靈芝幾丁質(Sacchachtin)之製備 39 3.3.2 靈芝幾丁聚醣(Sacchachitosan)製備 39 3.3.3 產率之測量 40 3.3.4 灰份之測量 40 3.3.5 去乙醯化程度之測定 40 3.3.6 分子量之測定 42 3.3.7 SEM表面結構觀察 43 3.3.8 抗菌測試 43 3.3.9 統計方法 44 第四章 結果與討論 45 4.1 利用不同PH值條件下對漂白之速率影響 45 4.2 靈芝子實體殘渣之衍生物產率測試 46 4.3 SACCHACHITIN物性分析 47 4.3.1 SEM之表面結構觀察 47 4.3.2 ATR-IR之定性分析觀察 48 4.3.3 13C CP-MAS NMR之定性分析觀察 51 4.3.4 分子量之測定 52 4.4 SACCHACHITOSAN物性分析 52 4.4.1 SEM之表面結構觀察 52 4.4.2 ATR-IR之定性分析觀察 52 4.4.3 13C CP-MAS NMR之定性分析觀察 53 4.4.4 PVSK膠體滴定法之去乙醯程度測量 54 4.4.5分子量之測定 55 4.4.6抑菌測試 56 第五章 結論 57 參考文獻 58 附錄 98
參考文獻 [1] 劉瓊淑,幾丁質、幾丁聚醣及相關酵素之特性與應用,食品工業, 1994,26(1):26-37。 [2] Knorr, D., Recovery and utilization of chitin and chitosan in food processing waste managrment. Food Technol, 1991. 45:114-122 [3] White, S. A., Farina, P. R., and Fulton, I., Production and isolation of chitosan from mucor rouxii. Appl. Environ. Microbiol, 1979. 38:323-328 [4] Tan, S. C., Tan, T. K., Wong, S. M., and Khor, E., The chitosan yield of zygomycetes at their optimum harvesting time. Carbohydrate polym, 1996. 30:239-242 [5] 濱口陽吉;木村三雄 (陽世益譯),螃蟹革命,青春出版社,1991, p.25。 [6] Sone, Y., Okuda, R., Wada, N., Kishida, E., and Misaki, A., Structures and antitumor activities of the polysaccharides isolated from fruiting body and the growing culture of mycelium of Ganoderma lucidum. Agric.Biol.Chem, 1985. 49(9): 1641-2653 [7] Su, C. H., Sun, C. S., Juan, S. W., Hu, C. H., Ke, W. T., and Sheu, M. T., Fungal mycelia as the source of chitin and polysaccharides and their applications as skin substitutes. Biomaterials, 1997. 18:1169-1174 [8] James, W. L., David, W. T., Producing proteins in transgenic plants and animals. Current Opinion in Biotechnol, 2001. 12: 411-418 [9] 林玫秀,零芝子實體殘渣衍生物的抗菌活性之研究,私立台北醫學大學醫學研究所碩士論文,2001。 [10] Corner, E. J. H., Ad Polyporaceae 1. Amauroderma and Ganoderma. Nova Hedwigia Beih, 1983. 75:1~182 [11] 林哲聖,靈芝在深層培養之研究,國立中興大學食品科學研究所 碩士論文,1990。 [12] 蔡宗統,台灣的靈芝,科學月刊,1989,20(6):424-426。 [13] 林志彬,靈芝的現代研究,北京醫科大學出版社,2001,p.14-31。 [14] 王伯徹;陳啟楨,靈芝,常見食藥用菇類介紹,1994,p.7-10。 [15] Masao, H., Chieko, I., Tsutomu, F., and Motoos, S., Ganoderic acids T、S and R,new triterpenoids from the cultured mycelia of Ganoderma lucidum. Chem.Pharm.Bull, 1986. 34(5):2282-2285 [16] Lin, C. N., Tome, W. p. and Won, S. J., Novel cytotoxic principles of Formosan Ganoderma lucidum. Journal of Natural Products, 1991. 54(4):998-1002 [17] Su, C. H., Hsu, J. J., Tung, T. C., Identification of species in the genus Ganoderma on patterns of triterpenoids by TLC scanner. J.Chinese Oncol.Soc, 1988. 4:9-11 [18] Morigiwa, A., Kitabatake, K., Fujimoto, Y. and Ikekawa, N., Angiotensin converting enzyme-unhibitory triterpenes from Ganoderma lucidum . Chem.Pharm.Bull, 1986. 34:3025-3028 [19] Kohda, H., Tokumoto, W., Sakamoto, K., Fujii, M., Hirai, Y., Yamasaki, K., Komoda, Y., Nakamura, H., Ishihara, S., and Uchida, M., The biological active constituents of Ganoderma lucidum (Fr.).Histamine release-inhibitory triterpenes. Chem.Pharm.Bull, 1985. 33:1367-1374 [20] Hikino, H., Ishiyama, M., Suzuki, Y., and Konno, C., Mechanisms of hypoglycemic activity of ganoderan B: a glycan of Ganoderma lucidum fruit bodies. Planta Medica, 1989. 55(5):423-8 [21] Komoda, Y., Shimizu, M., Sonoda, Y., and Sato, Y., Ganoderic acid aand its derivatives as cholesterol synthesis inhibitors. Chem.Pharm.Bull, 1985. 37(2):531-533 [22] 董一致;蘇慶華,靈芝神奇嗎?談靈芝特有之三?類成分,健康 世界,1988,35:72-74。 [23] Su, C. H., Taxonomy and physiologically active compounds of Ganoderma-A-review. 北醫學報, 1991. (20):1-16 [24] Kino, K., Yamashita, K., Watanabe, J., Tanaka, S., Ko, K., Shimizu, K., and Tsunoo, H., Isolation and characterization of a new immunomodulatory protein,ling zhi-8,from Ganoderma lucidum. J. Biol. Chem, 1989. 264(1):472-478 [25] Haak-Frendscho, M., Kino, K., Sone, T., and Jardieu, P., Ling zhi-8:a novel T cell mitogen indues cytokine production and upregulation of ICAM-1 expression. Cellular Immunology, 1993. 150(1):101-113 [26] Peberdy, J. F., Fungal cell walls-a review in “Biochemistry of cell walls and membranes in fungi”. Spring-Verlag,Berlin, 1989. 5-30 [27] 水野 卓;川合正允(賴慶亮譯),菇類的化學•生化學,國立編譯 館,1997。 [28] Hikino, H., Konno, C., Mirin, Y., and Hayashi, T., Isolation and hypoglycemic activity of ganoderans A and B, glycans of Ganoderma lucidum fruit bodies. Planta Medica, 1985. (4):339-340 [29] Hiroshi, H., and Takashi, M., Hypoglycemic actions of some heteroglycans of Ganoderma lucidum fruit bodies. Planta Medica, 1989. 55(4):385 [30] Seung, Y. L., and Hee, M. R., Cardiovascular wffects of mycelium extract of Ganoderma lucidum:Inhibition of sympathetic outflow as mechanism of its hypotensive action. Chem.Pharm.Bull, 1990. 38(5):1359-1364 [31] 陳大為;黃壤基;李旭生,靈芝對體外培養之口腔癌細胞的毒 殺效應,中華醫誌,1991,48:54-58。 [32] Zha ng, K., and Howard, R. P., Influence of polysaccharides on neutrophil function:specific antagonists suggest a model for cooperative saccharide-associated inhibition of immune complex-triggered superoxide production. J. of Cellular Biochemistry, 1994. 56:225-235 [33] Miyazaki, T., and Nishijima, M., Studies on fungal polysaccharides.XXVII.Structural examination of a water- soluble,antitumor polysaccharide of Ganoderma lucidum. Chem.Pharm.Bull, 1981. 29(12):3611-3616 [34] 鄭惠華;董一致;董大成,人工栽培之靈芝Ganoderma lucidum 萃取物之抗腫瘤作用III口服靈芝萃取亦對人體內T細胞亞群之影 響,中華癌症醫學會誌,1985,1(4):1-10。 [35] 黃雪芳;劉柯俊;管育慧;董光世;蘇慶華;董大成,口服靈 芝菌絲培養液之抗癌人工轉移作用,中華癌症醫學會誌,1989,5 (1):10-15。 [36] Lieu, C. W., Lee, S. S., and Wang, S. Y., The effect of Ganoderma lucidum on differentiation in leukemic U937 cells.Anticancer Research, 1992. 12(4):1211-1215 [37] Liu, F., Ooi, V. E. C., and Chang, S. T., Free radical scavenging activitives of mushroom polysaccharide extracts. Life Science, 1997. 60:763-771 [38] Ruiz-Herria, J., The distribution and quantitative importance of chitin in fungi. In:Proceedings of The First International Conference on Chitin and Chitosan. Ed. By. Muzzarelli, R. A., and Pariser, E. R. MIT Sea Grant Program, Cambridge, Mass, 1978. p:11-12 [39] 李遠豐,蟹殼膠特性應用及生產技術,生物產業,1998,9(1):27-37。 [40] Knorr, D., Use of chitinous polymers in food -- a challenge for food research and development. Food Tech, 1984. 38(1):85-97 [41] Su, C. H., Sun, C. S., Juan, S. W., Ho, H. O., Hu, C. H., and Sheu, M. T., Development of fungal mycelia as skin substitutes:effects on wound healing and fibroblast. Biomaterials, 1999. 20(1):61-8 [42] Sandford, P. A., Chitosan:commercial uses and potential applications.in:Chitin and chitosan, Proceedings of the Fourth International Conference on Chitin and Chitosan. Ed. By.Skjak-Braek, G., Anthonsen, T., and Sandford, P., Elsevier Applied Sci. Publishers, New York, 1988. p:51-69 [43] 王偉;秦紋;李素清;薄淑琴,甲殼素分子量,應用化學,1991, 8(6):85-87 [44] 林俊煌,不同去乙醯程度之幾丁聚糖的流變性質與鏈柔軟度、膜之物理特性的關係,國立台灣海洋大學水產食品科學研究所碩士論文,1992。 [45] Muzzarelli, R. A. A., and Rocchetti, R., Determination of the degree of acetylation of chitosans by first derivative ultraviolet spectrophotometry. Carbohydr.Polym, 1985. 5:461-472 [46] Roberts, G. A. F., Chitin chemistry. The MacMillan Press. London, 1992. [47] Poulicek, M., Voss-Fougart, M, F. , and Jeuniaux, C., Chitinoproteic complexs and mineralization in Mollusk skeletal Structures. in:Chitin in Nature and Technology, Proceedings of the Third International Conference on Chitin and Chitosan. Ed. By. Muzzarelli, R. A. A., Jeuniaux, C., and Gooday, G. W., Plenum Press, New York, 1985. p:7-12 [48] 張玉權,草蝦頭中幾丁質類產品的製備方法、理化性質及應用,國立台灣大學農業化學研究所碩士論文,1987。 [49] 戴明志,以氧化還原降解法從不同來源幾丁聚醣製備幾丁寡醣之探討,國立台灣海洋大學食品科學系碩士論文,1999。 [50] Niederhofer, A., Muller, B. W., A method for direct preparation of chitosan with low molecular weight from fungi. Eur J Pharm Biopharm, 2004. 57:101-105 [51] Bartnicki-Garcia, S., The biochemical cytology of chitin and Chitosan synthesis in fungi.In: Chitin and Chitosan: sources,chemistry,biochemistry,physical properties and application. Ed. By. Skjak-braek, G., Anthosen, T., and Sandford, P. Elserrier Applied Science, London, 1988. p:23-35 [52] Gooday, G. W., Control and inhibition of chitin synthesis in fungi and nematodes.In: Chitin and Chitosan: sources,chemistry,biochemistry,physical properties and application. Ed. By. Skjak-braek, G., Anthosen, T., and Sandford, P. Elserrier Applied Science, London, 1989. p:13-20 [53] Austin, P. R., Brine, C. J., Castle, J. E., and Zikakis, J. P., Chitin:new facets of research. Science.212, 1981. 15:749-753 [54] Aiba, S., Izumi, M., Minoura, N., and Fujiwara, Y., Studies on chitin.2.preoaration and properties of chitin membrane. Carbohydr. Polym, 1985. 5:285-289 [55] Tokura, S., Regeneration of α-chitin and its biodegradability.in Asia-Pacific Chitin and Chitosan Symposium. Universiti Kebangasaan Malaysia, Bangi, Malaysia, 1994. [56] 江晃榮,生物高分子(幾丁質、膠原蛋白)在食品工業上的應用,原料應用,1998,150(6):19-25。 [57] Chui, V. W. D., Mok, K. W., Ng, C. Y., Luong, B. P., Ma, K. K., Removal and recovery cooper(II),Chromium(III),and nikel(II)from solution using crude chrimp chitin packed in small columns. Environment International, 1996. 22:463-468 [58] Shahidi, F., Arachchi, J. K. V., and Jeon, Y. J., Food applications of chitin and chitosans. Food Sci.technol, 1999. 10:37-51 [59] 方紹威,幾丁質與幾丁聚醣在廢水處理、生化、食品和醫藥上之研究發展現況,藥物食品檢驗局調查研究年報,1990,8:20-30。 [60] Lepri, L.,and Desideri, P. G., Separation and identification of water-soluble food dyes by ion-exchange and soap thin-layer chromatography. J. chromatogr, 1978. 161:279-286 [61] Nishimura, K., Nishimura, S., Seo, H., Nishi, N., Tokura, S., and Azuma, I., Effect of multipoprous microspheres derived from chitin on the activation of mouse peritoneal macrophages. Vaccine, 1987. 5:136 -140 [62] Suzuki, S., Okawa, Y., Okura, Y., Hashimoto, K., and Suzuki, M., Proceedings of the second international conference on chitin and chitosan. Sapporp, Japan, 1982. p:210-212 [63] Deuchi, K., Kanauchi, O., Imasato, Y., and Kobayshi, E., Effect of the viscosity or deacetylation degree of chitosan on fecal fat excreted from rats fed on high-fat diet. Bioscience, Biotechnology & Biochemistry, 1995. 59(7):1211- 1216 [64] Fukasawa, M., Abe H., Masaoka T., Orita H., Horikawa H., Campeau, J. D., and Washio, M., The hemostatic effect of deacetylated chitin membrane on peritoneal injury in rabbit model. Surgery Today, 1992. 22(4):333-8 [65] 柯天來;彭光耀;江正陽;傅鍔;沈一慶,幾丁聚醣:新的牙周組織工程材料,中華牙周醫誌,2002,7(2):111。 [66] 蘇慶華,靈芝的抗菌作用,健康靈芝,2004,p.24-27。 [67] Frysh, H., Bowles, W. H., Baker, F., Rivera-Hidalgo, F., and Guillen, G., Effect of pH on hydrogen peroxide bleaching agents. J Esthet Dent, 1995. 7(3):130-3 [68] 翁家瑞,食品添加物,食品衛生與安全,2002,p.320-321。 [69] 呂世源,奈米新世界,科學發展,2002,p.4-7。 [70] 林志彬,靈芝的現代研究,北京醫科大學出版社,2001,p.157-205。 [71] Heux, L., Brugnerotto, J., Desbrieres, J., Versali, M. –F., and Rinaudo, M., Solid state NMR for degree of acetylation of chitin and chitosan. Biomacromolecules, 2000. 1: 746-751 [72] Kittur, F. S., Kumar, A. B. V., and Tharanathan, R. N., Low molecular weight chitosans-preparation by depolymerization with Aspergillus niger pectinase, and characterization. Carbohydr. Res, 2003. 338: 1283-1290 [73] Wu, T., Zivanovic, S., Draughon, F. A., and Sams, C. E., Chitin and chitosan-value-added products from mushroom waste. J. Agric. Food Chem, 2004. 52: 7905-7910 [74] Shimahara, K., Takiguchi, Y., Kobayashi, T., Uda, K., and Sannan, T. , Screening of mucoraceae strains suitable for chitosan production. In Chitin and Chitosan. Ed. By. Skjak-Braek, G., Anthonsen, T., Sanford, P., Elsevier, Lodon, 1989. p: 171-178 [75] No, H. K., Lee, S. H., Park, N. Y., and Meyers, S. P., Comparison of physicochemical, binding, and antibacterial properties of chitosans prepared without and with deproteinization process. J. Agric. Food Chem, 2003. 51: 7659-7663.

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系統識別號 U0007-1704200714542195
論文名稱(中文) 去乙醯幾丁聚醣之非對稱性膜滲透膠囊之製備與特性解析
論文名稱(英文) Characterization of Osmotic Capsule with Asymmetric Membrane Using Chitosan
校院名稱 臺北醫學大學
系所名稱(中) 藥學研究所
系所名稱(英) Graduate Institute of Pharmacy
學年度 93
學期 2
出版年 94
研究生(中文) 王聖希
學號 m301091011
學位類別 博士
語文別 英文
口試日期
論文頁數 107頁
口試委員 指導教授-許明照教授
關鍵字(中) 去乙醯幾丁聚醣
非對稱性膜
滲透膠囊
關鍵字(英) chitosan
asymmertric membrane
osmotic capsule
學科別分類
中文摘要 去乙醯幾丁聚醣之非對稱性膜滲透膠囊之製備與特性解析 中 文 摘 要 本研究主要目的是要以去乙醯幾丁聚醣(Chitosan)來當作非對稱性膠囊之材質,設計合適的滲透性控釋膠囊。非對稱性膜在膠囊化處理後可改善滲透性幫浦難溶性藥物釋放的情形。 膠囊殼乃是以交聯的方式膠化所產生,先將模具浸入含有聚合物的包覆溶液(coating solution),再浸入到淬鍊溶液(quenching solution),而在膜具上形成具有非對稱結構之膠囊殼。選用不同的膜衣材質,包覆溶液,浸漬溶液和浸漬時間來製作並評估非對稱結構之膠囊殼之特性。 滲透壓實驗中,薄膜釋藥孔之存在由包覆葉綠素的膠囊緩緩釋出葉綠素而得到證實。選用不同溶解度的藥物來評估對稱性膠囊的控釋效果。結果顯示藥物從非對稱性膜膠囊的釋放會因藥物的溶解度增加而增加,藥物的溶出速率與藥物溶解度成線性相關。在實用的觀點下考量製作難易度及通透效果後,選取以去乙醯幾丁聚醣與三磷酸鹽交聯三十分鐘這組膠囊為試驗膠囊。膠囊的通透率經計算而得 1.40*10-6 cm2/h-atm 於 攝氏37.8度,因為難溶性藥物felodipine 及 nifedipine不能提供足夠的滲透壓動力,於是在處方中添加助溶劑SLS 或添加助懸劑 HPMC 以增加藥物釋放。經計算而得之複回歸方程式: YF(drug max released %)= 32.48367-1.04456* F+0.14909 * H+ 0.23256 * S(mg) YN(drug max released %)= 30.99458-0.95958 * N+0.12802 * H+0.21321 * S(mg) 最後,我們成功地設計出以去乙醯幾丁聚醣與三磷酸鹽 (TPP)交聯之非對稱性滲透膠囊。
英文摘要 Characterization of Osmotic Capsule with Asymmetric Membrane Using Chitosan ABSTRACT The major purpose of the study was to devise a suitable osmotic capsule for drug release. In this study, chitosan was utilized as the matrix of asymmetric membrane capsules. Asymmetric membrane offering an improved osmotic pump effect was used to release poorly water-soluble drug in a control manner after encapsulation. The capsule wall was composed of membrane produced by gelation process, controlled by the ionic cross-linking reaction between protonated chitosan and ionized triphosphate (TPP). Asymmetric membrane was formed on stainless steel mold pins by dipping the mold pins into a coating solution containing a polymeric material followed by dipping into a quenching solution. The factors influenced the prosperities of the capsule membrane, such as the molecular weight of chitosan、the solvent、the dipping solution and the dipping time were investigated in view of the drug release. In situ formation of a delivery orifice in the thin membrane was proven by observation of a jet stream of chlorophyll being released from the capsule. Drugs with different solubility were selected as the model to demonstrate that the controlled release characteristics can be manipulated by the design of polymeric capsule coating an asymmetric membrane. The results showed that the release rate from asymmetric membrane capsule increased with the solubility for drugs. A correlation between drug solubility and the initial drug release rate calculated from the slope of the drug release profile was verified to be linear. From the practical viewpoint it is useful to estimate selectivity of the capsules towards the difficulty of production and permeability. The model capsule made of chitosan 500 cross-linked with triphosphate. Permeability across the selected asymmetric membrane of the C500/TPP30 capsule was determined to be 1.40*10-6 cm2/h-atm at 37.8OC for drugs with water solubilities in a moderate to high range. The poorly water-soluble drug, felodipine and nifedipine, were unable to create a sufficient osmotic effect on activating the release of drug. Solubilization either by the addition of the solubility enhancer, SLS, or by a solid dispersion with HPMC could increase the solubility of nifedipine to a sufficient extent to activate drug release. It was found that HPMC further interacted with SLS to synergistically increase the maximal percent of release amount and the release rate of felodipine and nifedipine. The multi regression formulations of felodipine and nifedipine were “YF( drug max released %) =32.48367-1.04456*F+0.14909*H+0.23256*S(mg)” and“YN(drug max released%) =30.99458-0.95958*N+0.12802*H+0.21321*S(mg)”, respectively. Consequently, we successfully designed the osmotic capsule with asymmetric membrane which was made of chitosan and cross-linked with TPP.
論文目次 English Abstract 13 Chinese Abstract 15 1. Introduction 17 1.1 The background of research 17 1.2 osmotic pump 18 1.2.1. Principles of osmosis 18 1.2.2. History of osmotic pump 20 1.2.2.1. The Rose-Nelson pump 20 1.2.2.2. Higuchi-Leeper pump 24 1.2.2.3. Higuchi-Theeuwes pump 25 1.2.2.4. Elementary osmotic pump (EOP) 26 1.2.3. Asymmetric membrane 32 1.3. Chitosan characteristic 32 1.3.1 Degree of deacetylation 33 1.3.2 Molecular weight 34 1.3.3. Application of chitosan in pharmaceutics 35 1.3.4. Classified of chitosan capsule 36 1.3.4.1. Chitosan hard capsule 36 1.3.4.2. Chitosan alginate capsule 37 1.4 Cross-linked membrane 38 1.5.The motive of the research 39 1.6 The profiles of the design 39 2. Experimental 40 2.1. Materials 40 2.2. Apparatus 41 2.3. Characteristic of chitosan 42 2.3.1 Molecular weight 42 2.3.2 Viscosity 42 2.3.3 Degree of deacetylation 42 2.4. Solvent 43 2.5. Quenching solution 44 2.6. Pure chitosan capsule 44 2.7. Chitosan/multivalent phosphates capsule 44 2.8. Solubility 46 2.9. Validation of assay model drugs 46 2.10. Release test 47 2.11. Osmotic pump capsule preparation 47 2.12. Theoretical considerations 50 3. Results and discussion 52 3.1. Characteristic of chitosan 52 3.1.1. Molecular weight 52 3.1.2 Viscosity 53 3.1.3. Degree of deacetylation 53 3.1.4. Solvent 54 3.2. Capsule preparation 56 3.2.1. Pure chitosan capsules 58 3.2.2. Chitosan/multivalent phosphates capsules 59 3.3. Physical characteristics of optimal chitosan capsules 3.4. Validation of assay method 64 3.5. In vitro drug release profiles 77 3.6. The permeability of chitosan/TPP and other chitosan/multi phosphates capsules 82 3.7. Release test of poor soluble drug 85 3.8. Validation of Dissolution Method 86 4. Conclusions 95 5. References 96 Table 1 Osmotic pressure for concentrated solutions of excipients 21 Table 2 Evolution of osmotic pump 28 Table 3 Elementary osmotic pump patents covering the use of various membranes 29 Table 4 Elementary osmotic pump patents covering design modifications 29 Table 5 Patent on some drugs formulated in the form of elementary osmotic pump 30 Table 6 Some commercially marketed products 31 Table.7 Chitin derivatives and their proposed uses 35 Table 8 Chitosan 100 coating solution formulations 43 Table 9 Quenching solution formulations 44 Table 10 Pure chitosan capsule formulations 45 Table 11 Chitosan/TPP capsule formulations 45 Table 12 Chitosan500/multivalent phosphates capsule formulations 46 Table 13 Design of chitosan osmotic capsules filled with Felodipine/HPMC/SLS 48 Table 14 Design of chitosan osmotic capsules filled with Nifedipine/HPMC/SLS 49 Table 15 Chitosan degree of deacetylation (DD) 54 Table 16 Chitosan morphology 57 Table 17 Characteristic profiles of chitosan capsule 63 Table 18 The solubility of drugs in water at 37.8℃ (n=3) 84 Table 19 Permeability of C500/multivalent phosphates capsules 85 Fig. 1. Profile of Pfeiffer’s osmosis experiment 19 Fig. 2. Rose-Nelson pump 21 Fig. 3. Pharmetrix pump 23 Fig. 4. Higuchi-Leeper pump 24 Fig. 5. Higuchi-Theeuwes pump 25 Fig. 6. Elementary osmotic pump 27 Fig. 7. The future of Alza’s OROS® system 28 Fig. 8. Structures of cellulose, chitin and chitosan. 33 Fig. 9. Chitosan capsules 37 Fig. 10. Membrane classification 38 Fig. 11. Chitosan molecular weights 52 Fig. 12. Chitosan viscosity 53 Fig. 13. Titration for chitosan degree of deacetylation 54 Fig. 14. Chitosan 100 dissolve in acetic/alcoholic solution (1:1) 55 Fig. 15. Chitosan 100 dissolve in acetic aqueous solution (3%w/w) 56 Fig. 16. Chitosan morphology (A) Chitosan 10 (B) Chitosan 100 (C) Chitosan 500 (D) Chitosan 1000 57 Fig. 17. Morphology of chitosan capsules 58 Fig. 18. Scanning electron micrographs of chitosan 500 membrane without quenching) formulation (A) dense region (outer layer) at 200 x magnification(B) cross-section at 1000 x magnification 59 Fig. 19. Morphology of chitosan/TPP capsules 60 Fig. 20. Morphology of chitosan/TPP capsules 60 Fig. 21. Scanning electron micrographs of chitosan membrane(quenching 15mins ) formulation cross-section at 200 x magnification(A)chitosan 10(B)chitosan100 (C)chitosan 500 (D)chitosan1000 61 Fig. 22. Scanning electron micrographs of Chitosan500 membrane formulation cross section at 200 X magnification (quenching 30 mins) 61 Fig. 23. Scanning electron micrographs of chitosan membrane formulation cross-section at 200 x magnification(A) C500/15Phos (B) C500/30Phos (C) C500/15Pyro (D) C500/30Pyro 62 Fig. 24. Photograph of asymmetric membrane C500/30TPP with chlorophyllin in water. (A)After 30 mins (B)After 60 mins(C)After 3hours(D)In NaCl 6%(w/w) solution(1 day) 64 Fig. 25. Intraday accuracy and precision of Chlorpheniramine 65 Fig. 26. Interday accuracy and precision of Chlorpheniramine 66 Fig. 27. Intraday accuracy and precision of Diltiazem 67 Fig. 28. Interday accuracy and precision of Diltiazem. 68 Fig. 29. Intraday accuracy and precision of Naproxen 69 Fig. 30. Interday accuracy and precision of Naproxen 70 Fig. 31. Intraday accuracy and precision of Pyridoxine. 71 Fig. 32. Interday accuracy and precision of Pyridoxine. 72 Fig. 33. Intraday accuracy and precision of Felodipine 73 Fig. 34. Interday accuracy and precision of Felodipine 74 Fig. 35. Interday accuracy and precision of Nifedipine 75 Fig. 36. Interday accuracy and precision of Nifedipine 76 Fig. 37. Chlorphenamine release profiles 78 Fig. 38. Diltiazem release profiles 78 Fig. 39. Naproxen release profiles 79 Fig. 40. Pyridoxine release profiles 79 Fig. 41. Felodipine release profiles 80 Fig. 42. The release profiles of chitosan capsules formulation C500,C500/TPP15 and C500/TPP30 in (A) Chlorpheniramine (B)Diltiazem (C)Naproxen (D)Pyridoxine (E)Felodipine 81 Fig. 43. Release profiles of formulation (A)C500/TPP15 (B) C500/TPP30 81 Fig. 44. Chitosan 500 capsules Linear relationship between the release rate and the square of solubility divided by molecular weight of the drug. (A)C500/TPP15 (B)C500/TPP30 83 Fig. 45. Release profiles of Felodipine in 1%Tween 80 solutions Felodipine (F)was made by HPMC(H) and physical mixed with SLS(S) 86 Fig. 46. Release profiles of Felodipine in 1%Tween 80 solutions Felodipine (F)was made by HPMC(H) and physical mixed with SLS(S) 87 Fig. 47. Release profiles of Nifedipine in 1%Tween 80 solutions Nifedipine (N)was made by HPMC(H) and physical mixed with SLS(S) 88 Fig. 48. Release profiles of Nifedipine in 1%Tween 80 solutions Nifedipine (N)was made by HPMC(H) and physical mixed with SLS(S) 89 Fig. 49. Release profiles of Nifedipine in 1%Tween 80 solutions Nifedipine(F) was made by HPMC(H) and physical mixed with SLS(S) 90 Fig. 50. Release profiles of Nifedipine in 1%Tween 80 solutions Nifedipine(F) was made by HPMC(H) and physical mixed with SLS(S) 91 Fig. 51. Release profiles of Nifedipine in 1%Tween 80 solutions Nifedipine(F) was made by HPMC(H) and physical mixed with SLS(S) 93 Fig. 52. The relationship among max released ratio/Felodipine/ HPMC 3D profiles (SLS amount was limited) 94 Fig. 53. The relationship among max released ratio/Nifedipine/ HPMC 3D profiles (SLS amount was limited) 95 Diagnostics Case Statistics A Felodipine 102 Diagnostics Case Statistics B Nifedipine 105
參考文獻 References 1. R. K.Verma, D. M. Krishna, S. Garg, Formulation aspects in the development of osmotically controlled oral drug delivery systems, Journal of Controlled Release 79 (2002) 7-27 2. R. K. Verma, B. Mishra, S. Garg, Osmotically Controlled Oral Drug Delivery, Drug Development and Industrial Pharmacy, 26, (2000) 695—708 3. W. A, Weller PJ. Handbook of pharmaceutical excipients, 2nd ed., Pharmaceutical Press, London, pp. (1994) 428-429 4. S. Rose, J. F. Nelson, A continuous long-term injector, Journal of Exp. Biology 33 (1955) 415 5. A. L. Athayde, and R. A. Faste, Osmotic infusion device, U.S. Patent 5,169,390 (1990) 6. G. Santus, Osmotic drug delivery, Journal of Controlled Release 35 (1995) 1-21 7. F. Theeuwes, and A. D. Ayer, Osmotic system having laminar arrang- ement for programming delivery of active agent, U.S. Patent 4,008,719 (1977) 8. S. M. Herbig, J. R. Cardinal, R.W. Korsmeyer, K.L. Smith, the polymer was equally important as that of the Asymmetric membrane tablet coatings for osmotic drug osmotic action. delivery, J. Controlled Release 35 (1995) 127—136. 9. A. G. Thombre, J. R. Cardinal, A. R. DeNoto, S. M. Herbig, and K. L. Smith, Asymmetric membrane capsules for osmotic drug delivery I. Development of a manufacturing process, Journal of Controlled Release 57 (1999) 55-64 10. A. G. Thombre, J. R. Cardinal, A. R. DeNoto, and D. C. Gibbes, Asy- mmetric membrane capsules for osmotic drug delivery II. In vitro and in vivo drug release performance, Journal of Controlled release (2002) 12-18 11. R. Rautenbath, Membrane process, John Willey and Stones, New York, Chapter 2, (1989) 18-47 12. F. Theeuwes, R. J. Saunders, and W. S. Mefford, Process for forming outlet passageways in pills using a laser, U.S. Patent 4,088,864 (1978) 13. W. J. Curatolo, Dispensing devices powered by lyotropic liquid crystals, U.S. Patent 5,108,756, April 28, 1992. 14. W. J. Curatolo, Dispensing devices powered by lyotropic liquid crystals, U.S. Patent 5,030,452, Jan. 12, 1989. 15. A. G. Thombre, Delivery device having encapsulated excipients, U.S. Patent 5,697,922, Dec. 6, 1997 16. A. G. Thombre, A. R. DeNoto, D. C. Gibbes, Delivery of glipizide from asymmetric membrane capsules using encapsulated excipients, J. Controlled Release 60 (1999) 333—341. 17. K. Okimoto, R.A. Rajewski, V.J. Stella, Release of testosterone from an osmotic pump tablet utilizing(SBE)-7mβ-CD as both a solubilizing and an osmotic pump agent, J. lease granules in vitro and healthy subjects , Chem. Pharm.Controlled Release 58 (1999) 29—38. 18. A. D. Koparkar, S. B. Shah, Oral osmotic system for slightly soluble active agents, U.S. Patent 5,284,662, (1994) 8. 19. R. A. Muzzrelli, M. P. Peter, Chitin Handbook, Chapter 6 (1997) 20. N. V.Majeti, R. Kumar, A review of chitin and chitosan applications, Reactive & Functional Polymers 46 (2000)1—27 21. R. A. A. Muzzarelli (Ed.), Pergamon Press,Natural Chelating Polymers, (1973) 83 22. T. Kiang, J. Wen, H. W. Lim, K. W. Leong., The effect of the degree of chitosan deacetylation on the efficiency of gene transfection, Biomaterials 25 (2004) 5293-5301 23. R. A. A. Muzzarelli, C. Lough, M. Emanuelli, The molecular weight of chitosan studied by laser light scattering, Carbohydr. Res. 164 (1987) 433. 24. R. H. Chen, H. D. Hwa, Effect of molecular weight of chitosan with the same degree of deacetylation on the thermal, mechanical, and permeability properties of the prepared membrane, Carbohydrate Polymer 29 (1996) 353-358 25. N. V. Majeti, R. Kumar, A review of chitin and chitosan applications Reactive & Functional Polymers 46 (2000) 1—27 26. Y. Xu, Y. Du, Effect of molecular structure of chitosan on protein delivery properties of chitosan nanoparticles, International Journal of Pharmaceutics 250 (2003) 215-226 27. Y. Sawayanagi, N. Nambu, T. Nagai, Compressed tablets J. Kost (Ed.), Pulsed and Self-Regulated Drug Delivery, containing chitin and chitosan in addition to lactose or CRC Press, Boca Raton, 1990. potato starch, Chem. Pharm. Bull. 30 (1982) 2935. 28. 松本 隆幸¸ 東出 年弘, 公開特許公報, キトサン硬カプセル及ぶその製造法 (平成六年, 1994 )1-12 29. H.Tozaki, T. Fujita, T. Odoriba, Colon-specific delivery of R68070, a new thromboxane synthase inhibitor, using chitosan capsules: Therapeutic effects against 2,4,6-trinitrobenzene sulfonic acid-induced ulcerative colitis in rats, Life Sciences 64 (1999) 1155-1162 30. H. Tozaki, T. Odoriba , N. Okada , T. Fujita , A. Terabe , T. Suzuki , S. Okabe, S. Muranishi , A. Yamamoto, Chitosan capsules for colon- specific drug delivery: enhanced localization of 5-aminosalicylic acid in the large intestine accelerates healing of TNBS-induced colitis in rats, Journal of Controlled Release 82 (2002) 51—61 31. F. L. Mi, N. L. Her, C. Y. Kaun, T. Wong, S. Shyu, Chitosan tablets for controlled drug release of theophylline: effect of polymer drug wet or dry blending and anionic—cationic inter polymer, Journal of Applicated Polymer Science 66 (1997) 2495 32. Daly, Mary M, Keown. Chitosan alginate capsules. U.S.Patent 4,808,707. February 28, 1987 33. M. Li, N. Minoura, L. Dai, L. Zhang : Cross-linking techniques applied to asymmetric membranes, Macromolecular Material and Engineering 286(9) (Sep. 2001) 529-533 34. L. Hua, Z.H. Sun, Y. Leng, Z.Q. Hu, Continuous biocatalytic resolution of dl-pantolactone by cross-linked cells in a membrane bioreactor, Process Biochemistry 40 (2005) 1137—1142 35. C. Caoa, T. S. Chung, Y. Liu, R. Wang, K.P. Pramoda, Chemical cross-linking modification of 6FDA-2,6-DAT hollow fiber membranes for natural gas separation, Journal of Membrane Science 216 (2003) 257—268 36. 林山陽,膜衣包覆技術,九州出版社,眾光文化事業有限公司,第二、三章(1996) 37. R. H. Chen, H. D. Hua, Effect of molecular weight of chitosan with the same degree of deacetylation on the thermal, mechanical and permeability properties of the prepared membrane, Carbohydrate polymer 29 (1996) 353-358 38. Y. K. Lin, H. O. Ho, Investigations on the drug releasing mechanism from an asymmetric membrane-coated capsule with an in situ formed delivery orifice, Journal of Controlled Release 89 (2003) 57—69 39. F. L. Mi, S. S. Shyu, C. T. Chen, J. Y., Porous chitosan microsphere for controlling the antigen release of Newcastle disease vaccine: preparation of antigen-adsorbed microsphere and in vitro release Schoung, Biomaterials 20 (1999) 1603-1612

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系統識別號 U0007-1704200714562870
論文名稱(中文) 尼古丁乙醯膽素受體於台灣婦女乳癌組織之表現
論文名稱(英文) Nicotinic Acetylcholine Receptors Expression in Breast Cancer Tissues of Taiwanese Female Patients
校院名稱 臺北醫學大學
系所名稱(中) 醫學科學研究所
系所名稱(英) Graduate Institute of Medical Sciences
學年度 92
學期 2
出版年 93
研究生(中文) 譚家偉
學號 M102091001
學位類別 碩士
語文別 中文
口試日期
論文頁數 49頁
口試委員 指導教授-吳志雄
指導教授-何元順
關鍵字(中) 尼古丁乙醯膽素受體
尼古丁
乳癌
關鍵字(英) nAchRs
nicotine
NNK
tobacco-related carcinogen
Akt
MCF-7
MDA-MB-231
Breast cancer
學科別分類
中文摘要 中文摘要   目前流行病學的研究,仍無法確定吸煙是否會促進乳癌的發生。但研究顯示serine/threonine kinase Akt (protein kinase B) 的活化在癌症發生扮演一重要角色,Akt pathway的活化於肺部上皮細胞受到尼古丁(nicotine)及煙草特異致癌物4-(methylnitrosamino) -1-(3-pyridyl)-1- butanone (NNK) 誘導,於乳房細胞則受雌性激素(estradiol)所誘導。 這些研究提供了一個假設,即nicotine及NNK的暴露同樣能活化乳房細胞的Akt,進而導致乳房細胞增生(proliferation)。本研究的目的即在台灣乳癌婦女的乳房組織上,評估其尼古丁乙醯膽素受體(nicotinic acetylcholine receptors, nAchRs) 的表現,進而分析這些受體表現與臨床病理的關係。我們收集了十八位侵犯性管道癌 (invasive ductal carcinoma)及兩位原位癌(ductal cancinoma in situ)之乳房組織,以反轉錄聚合酶連鎖反應(Reverse Transcription-Polymerase Chain Reaction, RT-PCR)偵測乳癌組織、癌病變旁之正常組織以及人類乳癌細胞株(human breast cell lines)的nAchRs之cDNA表現。實驗顯示於二十例乳癌組織中,十九例顯示出有α9 nAchRs表現,十一例顯示有α10 nAchRs表現;於二十例正常之乳房組織中,十八例及九例顯示出含有α9及α10之nAchRs表現,其他nAchR次單元的表現則較不明顯。進一步比較nAchR在乳癌及正常乳房組織的表現強度,則α9與α10次單元於乳癌組織上的表現大部份均較正常組織上的表現強。且當α9與α10次單元同時表現於乳癌及正常組織時,該乳房病變的體積越大 (P=0.009)。以多變項直線模型進一步分析顯示,相較於其他次單元,乳癌細胞有α10之nAchRs表現為影響腫瘤大小的最主要因子。而乳癌細胞株MCF-7,則發現含有α1、α3、α5、α7、α9、α10 和β4 nAchRs之表現,而MDA-MB-231細胞株則只有α5及α9次單元表現。以上結果,對nicotine及NNK促使人類乳房細胞癌化及成長此一假說,提供了立足點,惟仍需進一步之細胞及動物實驗加以證明。
英文摘要 Abstract Epidermiological reports on the role of smoking in breast carcinogenesis are controversial. However, evidences indicate that rapid serine/threonine kinase Akt (Protein kinase B) activation play an important role of carcinogenesis, such pathway were induced by nicotine and the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)- 1-butanone (NNK) in lung epithelial cell and also by estradiol in breast cell. It provides the idea that nicotine and NNK exposure may activates Akt , resulting of cell proliferation in breast cell. Our objective of study is to evaluate the nicotinic acetylcholine receptors (nAchRs) expression in breast cancer tissues of Taiwanese female patients, and the relation of clinicopathologic features between such receptors expression is analyzed. We obtained human breast specimens from 18 patients with invasive ductal carcinoma and 2 patients with ductal carcinoma in situ. cDNA of nAchRs were studied by Reverse Transcription- Polymerase Chain Reaction (RT-PCR) in breast cancer specimens, normal breast tissue adjacent to the cancer and human breast cancer cell lines. α9-containing andα10-containing nAchRs were expressed in 19 and 11 of 20 breast cancer samples; they were also expressed in 18 and 9 of 20 normal breast samples adjacent to the cancer, respectively. Expression of other subunits of nAchR are not frequent. Compare with the intensity of nAchR band, most specimens showed significant expression of α9 and α10 subunits in breast cancer tissue relative to the corresponding normal tissue. Besides, there was a significant association between α9- containing andα10-containing nAchRs synchronous expression in both cancer and normal tissues with local tumor extent (P=0.009). Multivariate general linear model showed thatα10-containing nAchR expression in breast tumor cells is the most importart factor of tumor size among the other subunits. In breast cancer cell line MCF-7, α1, α3, α5, α7, α9, α10 andβ4-containing nAchRs were expressed, whereas onlyα5 andα9 subunits expression in MDA-MB-231 cell. Our data support the hypothesis that nicotine and NNK may contribute to the initiation and progression of human breast cancer, further in vitro and in vivo studies to confirm such hypothesis is required. Key words: nAchRs, nicotine, NNK, tobacco-related carcinogen, estradiol, Akt, MCF-7, MDA-MB-231, invasive ductal carcinoma, ductal carcinoma in situ
論文目次 目錄 縮寫表 英文摘要 中文摘要 第一章 前言 壹、研究目的 貳、文獻回顧 一、乳癌 (一)乳癌的流行病學 (二)乳癌的病理學分期 (三)乳癌的治療 (四)乳癌的危險因子 二、吸煙 (一)吸煙與癌症 (二)吸煙與乳癌的關係 三、尼古丁乙醯膽素受體 第二章 研究對象及實驗方法 壹、研究對象 一、研究對象 二、標本的收集 三、臨床資料的收集 貳、實驗方法 一、細胞的培養 二、反轉錄-聚合酶連鎖反應 三、統計分析 第三章 實驗結果 一、乳癌病患之臨床病理特性分析 二、nAchRs在乳房正常組織及乳房腫瘤之表現 三、nAchRs表現與臨床病理的關係 四、吸煙在nAchRs表現與臨床病理的關係上扮演的角色 五、nAchRs在乳癌細胞株的表現 圖目錄 Table 1 TNM clinical classification of breast cancer Table 2 Stage of breast cancer Table 3 Risk factors of breast cancer Figure 1 Structures of organic pulmonary carcinogens in tobacco smoke Table 4 Classification of cholinergic receptor Figure 2 Structure of nAchRs 附錄一 PCR之primers序列 Table 5 Patient Characteristics Table 6 nAchRs expression in normal and tumor breast tissues Figure 3 α9-containing nAchR expression in breast tissues Table 7 nAchR expression related to clinicopathologic features Figure 4 nAchRs expression in MCF-7 cell Figure 5 nAchRs expression in MDA-MB-231 cell Table 8 nAchRs expression in breast cancer cell lines 第四章 討論 參考文獻
參考文獻 參考文獻 1.Ries LAG, Eisner MP, Kosary CL, et al.(eds). SEER Cancer Statistics Review, 1973-1997, National Cancer Institute. NIH Pub. No. 00-2789. Bethesda MD,2000. 2.Jemal A and others. Cancer Statistics, 2002. CA Cancer J Clin. 2002;52: 23-47. 3.Krieger N. Social class and the black/white crossover in the age-specific incidence of breast cancer: a study linking census-derived data to population-based registry records. Am J Epidemiol, 1990;131(5): 804-14. 4.Singletary SE. Rating the risk factors for breast cancer. Ann Surg, 2003;237: 472-482. 5.Tobacco or health: a global status report. Geneva: WHO. 1-48. 6.Tobacco smoking. IARC monographs on the evaluation of the carcinogenic risk of chemicals to human (Vol 38). Lyon, France. IARC; 37-375. 7.Doll R. Cancers weakly related to smoking. Br Med J, 1996;52: 35-49. 8.Hoffmann D, Hoffmann I, El Bayoumy K. The less harmful cigarette: a controversial issue. a tribute to Ernst L. Wynder. Chem Res Toxicol, 2001; 14(7): 767-90. 9.Hecht SS. Tobacco carcinogens, their biomarkers and tobacco-induced cancer. Nat Rev Cancer, 2003;3(10): 733-44. 10.Heeschen C, Jang JJ, Weis M, Pathak A, Kaji S, Hu RS, Tsao PS, Johnson FL, Cooke JP. Nicotine stimulates angiogenesis and promotes tumor growth and atherosclerosis. Nat Med, 2001;7: 833-9. 11.Heeschen C, Weis M, Aicher A, Dimmeler S, Cooke JP. A novel angiogenic pathway mediated by non-neuronal nicotinic acetylcholine receptors. J Clin Invest, 2002;110(4):527-36. 12.Hecht SS. Tobacco smoke carcinogens and lung cancer. J Natl Cancer Inst, 1999;91(14): 1194-210. 13.Palmer JR, Rosenberg L. Cigarette smoking and the risk of breast cancer. Epidemiol Rev, 1993;15(1): 145-56. 14.Jee SH, Ohrr H, Kim IS. Effects of husbands'''' smoking on the incidence of lung cancer in Korean women. Int J Epidemiol, 1999;28(5): 824-8. 15.Lash TL, Aschengrau A. Active and passive cigarette smoking and the occurrence of breast cancer. Am J Epidemiol, 1999;149(1): 5-12. 16.Morabia A. Smoking (active and passive) and breast cancer: epidemiologic evidence up to June 2001. Environ Mol Mutagen, 2002;39(2-3): 89-95. 17.Hecht SS. Tobacco smoke carcinogens and breast cancer. Environ Mol Mutagen, 2002;39(2-3): 119-26. 18.Reynolds P., Hurley S, Goldberg DE, Anton-Culver H, Bernstein L, Deapen D, Horn-Ross PL, Peel D, Pinder R, Ross RK, West D, Wright WE, Ziogas A. Active smoking, household passive smoking, and breast cancer: evidence from the California Teachers Study. J Natl Cancer Inst, 2004;96(1): 29-37. 19.Shrubsole MJ, Gao YT, Dai Q, Shu XO, Ruan ZX, Jin F, Zheng W. Passive smoking and breast cancer risk among non-smoking Chinese women. Int J Cancer, 2004;110(4): 605-9. 20.Terry PD, Rohan TE. Cigarette smoking and the risk of breast cancer in women: a review of the literature. Cancer Epidemiol Biomarkers Prev, 2002;11(10): 953-71. 21.Band PR, Le ND, Fang R, Deschamps M, Carcinogenic and endocrine disrupting effects of cigarette smoke and risk of breast cancer. Lancet, 2002; 360(9339): 1044-9. 22.Li D., Zhang W, Sahin AA, Hittelman WN. DNA adducts in normal tissue adjacent to breast cancer: a review. Cancer Detect Prev, 1999;23(6): 454-62. 23.Kadohama N, Shintani K, Osawa Y. Tobacco alkaloid derivatives as inhibitors of breast cancer aromatase. Cancer Lett, 1993;75(3): 175-82. 24.Perera FP, Estabrook A, Hewer A, Channing K, Rundle A, Mooney LA, Whyatt R, Phillips DH. Carcinogen-DNA adducts in human breast tissue. Cancer Epidemiol Biomarkers Prev, 1995;4(3): 233-8. 25.Stoica GE, Franke TF, Wellstein A, Czubayko F, List HJ, Reiter R, Morgan E, Martin MB, Stoica A. Estradiol rapidly activates Akt via the ErbB2 signaling pathway. Mol Endocrinol, 2003;17(5): 818-30. 26.Sun M, Paciga JE, Feldman RI, Yuan Z, Coppola D, Lu YY, Shelley SA, Nicosia SV, Cheng JQ. Phosphatidylinositol-3-OH Kinase (PI3K)/AKT2, activated in breast cancer, regulates and is induced by estrogen receptor alpha (ERalpha) via interaction between ERalpha and PI3K. Cancer Res. 2001;61(16):5985-91. 27.Bacus SS, Altomare DA, Lyass L, Chin DM, Farrell MP, Gurova K, Gudkov A, Testa JR. AKT2 is frequently upregulated in HER-2/neu-positive breast cancers and may contribute to tumor aggressiveness by enhancing cell survival. Oncogene, 2002;21(22): 3532-40. 28.Arboleda MJ, Lyons JF, Kabbinavar FF, Bray MR, Snow BE, Ayala R, Danino M, Karlan BY, Slamon DJ, Overexpression of AKT2/protein kinase Bbeta leads to up-regulation of beta1 integrins, increased invasion, and metastasis of human breast and ovarian cancer cells. Cancer Res, 2003;63(1):196-206. 29.West KA, Brognard J, Clark AS, Linnoila IR, Yang X, Swain SM, Harris C, Belinsky S, Dennis PA. Rapid Akt activation by nicotine and a tobacco carcinogen modulates the phenotype of normal human airway epithelial cells. J Clin Invest, 2003;111(1): 81-90. 30.Hogg RC, Raggenbass M, BertrandD. Nicotinic acetylcholine receptors: from structure to brain function. Rev Physiol Biochem Pharmacol, 2003;147: 1-46. 31.Schuller HM, Plummer HK 3rd, Jull BA. Receptor-mediated effects of nicotine and its nitrosated derivative NNK on pulmonary neuroendocrine cells. Anat Rec, 2003;270A(1): 51-8. 32.Minna JD. Nicotine exposure and bronchial epithelial cell nicotinic acetylcholine receptor expression in the pathogenesis of lung cancer. J Clin Invest, 2003;111(1): 31-3. 33.Schuller HM, Orloff M. Tobacco-specific carcinogenic nitrosamines. Ligands for nicotinic acetylcholine receptors in human lung cancer cells. Biochem Pharmacol, 1998;55(9): 1377-84. 34.Astles PC, Baker SR, Boot JR, Broad LM, Dell CP, Keenan M. Recent progress in the development of subtype selective nicotinic acetylcholine receptor ligands. Curr Drug Targets CNS Neurol Disord, 2002;1(4): 337-48. 35.Olale F, Gerzanich V, Kuryatov A, Wang F, Lindstrom J. Chronic nicotine exposure differentially affects the function of human alpha3, alpha4, and alpha7 neuronal nicotinic receptor subtypes. J Pharmacol Exp Ther, 1997;283(2): 675-83. 36.Schuller HM, Jull BA, Sheppard BJ, Plummer HK. Interaction of tobacco-specific toxicants with the neuronal alpha(7) nicotinic acetylcholine receptor and its associated mitogenic signal transduction pathway: potential role in lung carcinogenesis and pediatric lung disorders. Eur J Pharmacol, 2000;393(1-3): 265-77. 37.Mei J, Hu H, McEntee M, Plummer III H, Song P, Wang HCR. Transformation of non-cancerous human breast epithelial cell line MCF 10A by the tobacco-specific carcinogen NNK. Breast Cancer Res Treat, 2003;79: 95-105. 38.Dimmeler S, Zeiher AM. Akt takes center stage in angiogenesis signaling. Circ Res, 2000;86(1): 4-5. 39.Chau NM, Ashcroft M. Akt2: a role in breast cancer metastasis. Breast Cancer Res, 2004;6(1): 55-7. 40.Zhu BQ, Heeschen C, Sievers RE, Karliner JS, Parmley WW, Glantz SA, Cooke JP. Second hand smoke stimulates tumor angiogenesis and growth. Cancer Cell, 2003;4(3): 191-6.

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系統識別號 U0007-1704200714562982
論文名稱(中文) 以流式細胞技術評估肌無力病患之免疫功能
論文名稱(英文) Immune Function Assessment of Myasthenia Gravis Patients Using Flow Cytometry
校院名稱 臺北醫學大學
系所名稱(中) 醫學科學研究所
系所名稱(英) Graduate Institute of Medical Sciences
學年度 93
學期 2
出版年 94
研究生(中文) 簡珮如
學號 M102091014
學位類別 碩士
語文別 中文
口試日期
論文頁數 118頁
口試委員 指導教授-施純明
關鍵字(中) 重症肌無力症
乙醯膽鹼接受體抗體
淋巴細胞亞群分布
細胞吞噬活性
自然殺手細胞
細胞凋亡
細胞週期
關鍵字(英) myasthenia gravis
acetylcholine receptor antibody
lymphocyte subpopulations
phagocytic activity
natural killer cell activity
apoptosis
cell cycle
學科別分類
中文摘要 肌無力症(Myasthenia gravis; MG)為器官專一的自體免疫疾病。在病人體內由於存有直接對抗肌膜上的〝乙醯膽鹼接受體(AchR)〞之抗體,以致神經興奮之乙醯膽鹼無法與受體相結合而出現肌肉無力的症狀。此種乙醯膽鹼接受體抗體 (AchRAb)即為引發肌無力症之致病因子。在85%的廣泛型肌無力症病人體內皆可測得此抗體。因此肌無力症之治療方式之ㄧ是以能降低血中乙醯膽鹼接受體抗體為主要目標。 本論文目前分析經由血漿分離術 (plasmapheresis)之20位肌無力症病人 (8男:12女)及19位正常志願者 (10男:9女),以流式細胞儀檢測其淋巴細胞亞群分布(lymphocyte subpopulations)、細胞吞噬活性(phagocytic activity)、自然殺手細胞毒殺能力(natural killer cell activity)、周邊血液單核細胞(peripheral blood mononuclear cells, PBMC)的細胞凋亡比例(apoptosis percentage)以及細胞週期(cell cycle)分布情形,同時和正常人作比對。以淋巴細胞免疫分型法(immunophenotyping)進行淋巴球亞群分布分析,發現肌無力症病患的自然殺手細胞(natural killer cell, NK)高於正常人約1.3倍 (p=0.052) ,特別是男性肌無力病患的自然殺手細胞高於女性約1.61倍 (p<0.050) ,然而男性肌無力病患B細胞 (B cells)較正常人低0.72倍 (p<0.050)。在幫助性T細胞 (T helper cells, Th)方面,單純肌無力無胸腺增生 (hyperplasia)也無胸腺瘤 (thymoma)較正常人、肌無力合併胸腺增生及肌無力合併胸腺瘤分別高1.26倍 (p <0.050) 、1.46倍 (p <0.050) 及1.49倍 (p <0.050) ;在胸腺瘤開刀後較無開刀的病人低0.64倍 (p <0.050) 。有使用藥物的病患較沒有使用藥物的病患在總T細胞(total T cells; T)下降約0.81倍 (p <0.050)。然而在單核球(monocytes)的吞噬活性分析,肌無力病人有較低的趨勢;但在顆粒球(granulocytes)的吞噬活性方面並無明顯差異。在肌無力症具有胸腺增生者較正常人、單純肌無力無胸腺增生也無胸腺瘤病患及肌無力合併胸腺瘤者,分別低0.97倍 (p <0.050) 、0.96倍 (p <0.050) 及0.96倍 (p <0.050)。女性病患、年齡超過40歲、病程小於兩年或非肌眼型的患者,NK細胞活性比正常人皆有上升之趨勢,然而若病患使用免疫抑制藥物之後NK細胞活性則明顯下降(p <0.050)。使用亞二倍體法(hypodiploidy analysis)分析肌無力病患PBMC的細胞凋亡比例較正常人高2.25倍 (p <0.050) ,特別是肌眼型病患較正常人高2.72倍 (p <0.050)。PBMC的細胞週期,S期在肌無力病患上升約3.24倍 (p <0.050) ,特別是肌無力女性病患較男性高3.14倍 (p <0.050)。而使用免疫抑制藥物之後肌無力病患的S期較正常人及未使用藥物病患分別高4.39倍 (p <0.050) 及2.73倍 (p <0.050) ; G0/G1期則較未使用藥物病患低0.92倍(p <0.050)。 利用血漿分離術治療肌無力病患,以降低病人體內乙醯膽鹼接受體抗體含量,特別是具有「呼吸窘迫之危象」的病患,可使病患在臨床症狀上快速獲得改善。肌無力病患在血漿分離術治療後,不但 T細胞、幫助性T細胞及Th/Ts ratio下降,而且B細胞、抑制性T細胞及NK細胞上升,但無顯著差異。單核球及顆粒球的吞噬活性方面則無明顯差異。在血漿分離術治療後,NK細胞毒殺力明顯降低。周邊血液單核細胞之細胞凋亡比例肌無力患者血漿分離術治療後明顯上升約2倍(p=0.021) ,以女性患者細胞凋亡比例上升較有統計意義。G0/G1 phase在治療後有下降之趨勢,S phase及G2/M phase則有上升之趨勢,女性病患細胞週期改變較為明顯,但無顯著差異。 綜合以上結果,利用流式細胞儀可快速評估肌無力病人的免疫功能。
英文摘要 Myasthenia gravis (MG) is an organ-specific autoimmune disease generally mediated by antibodies against the acetylcholine receptor (AChR) of skeletal muscle, characterized by fatiguable muscle weakness. This impairment is attributed to the presence of autoantibodies directed against the AChR. In 85% of MG patients had raised levels of AChR-specific antibodies by this approach. Therefore, it is an important issue to decrease AChR antibodies for treatment of MG patients. To assess the immune functions of MG patients after plasmapheresis, a total of 20 MG patients (M8: F12) and 19 normal volunteers (M10: F9) was collected and subjected for analysis of their lymphocyte subpopution, phagocytic activity, Natural killer cell activity, apoptosis percentage and cell cycle distribution of peripheral blood mononuclear cell(PBMC) using flow cytometry. Results of lymphocyte subset indicated that the NK cell proportion of MG patients was increased about 1.3 folds (p=0.052) while comparing with normal controls. Specially, MG male NK cells were higher than that of female for about 1.61 fold (p<0.050). In contrast, B cells of MG male were lower than that normal volunteers for about 0.72 fold. For T helper cells, the MG patients without hyperplasia and thymoma were higher than those of normal volunteers, MG patients with hyperplasia, MG patients with thymoma for about 1.26- (p<0.050), 1.46- (p<0.050), and 1.49-folds (p<0.050), respectively. Th cells proportion in MG patients with thymoma operation observed for 0.64 fold (p<0.050) lower than that without operation. A decrease of 0.81 fold (p<0.050) was observed in MG patients treated with drug than that without drug administration. Otherwise, the phagocytic activity of monocyte excerted a trend of decrease than the normal control. NK cell activity of MG patients with female, age over 40, duration two years or without ocular was likely higher than the normal controls but no statistical significantly. Nevertheless, the NK cell activity of immunosuppression- treated patients were significantly decreased than that without drug treatment. The PBMC apoptotic percentage of MG patients was 2.25 folds higher than that of normal controls (p<0.050), especially the ocular-MG patients were 2.72 folds higher than normal controls (p<0.050). For cell cycle analysis, there was a trend that the S phase was 3.24 fold (p<0.050) elevated in MG patients, especially the MG females were high than males for about 3.14 folds (p<0.050). The S phase of drug-treated MG patients was higher than that of the normal controls and the ones without drug treatment for about 4.39-(p<0.050), 2.73-folds (p<0.050). In addition, the G0/G1 phase in MG patients treated with drug was lower than the ones without drug treatment for about 0.92 fold (p<0.050). Double filtration plasmapheresis (DFP) is a useful methoud for reducing the amount of AChR antibodies which is able to improved the MG clinical condition. For lymphocyte subpopulation assay, the total T cells, Th cells, and Th/Ts ratio were decreased, whereas the B cells and NK cells were increased after DFP without statistical significance. After DFP, B cells (p<0.001), Th cells (p<0.010) and Th/Ts ratio (p<0.001) of healthy volunteers were increased, whereas Ts cells (p<0.010), NK cells and total T cells were decreased. Moreover, the apoptotic percentage of PBMC was increased for about two fold (p=0.021) in MG patients after DFP, especially in female patients. For analysis of cell cycle, G0/G1 phase decreased but S phase and G2/M phase increased in MG female. In conclusion, this study established a flow cytometric method which provides a verified assessment for the immune functions of MG patients.
論文目次 章節目錄 章節目錄………………………………………………………………Ⅰ 中文摘要………………………………………………………………Ⅱ 英文摘要………………………………………………………………Ⅴ 圖表目次………………………………………………………………Ⅶ 縮寫表…………………………………………………………………XⅡ 緒論…………………………………………………………………… 1 實驗材料與方法………………………………………………………19 結果……………………………………………………………………31 討論……………………………………………………………………41 未來實驗方向及展望…………………………………………………47 參考文獻………………………………………………………………49 表………………………………………………………………………59 圖………………………………………………………………………63 附錄………………………………………………………………… 102
參考文獻 Aarli JA, Stefansson K, Marton LS and Wollmann RL. Patients with myasthenia gravis and thymoma have in their sera IgG autoantibodies against titin. Clin. Exp. Immunol. 82: 284-8, 1990. Aarli JA. Late-onset myasthenia gravis. A changing scene. Arch Neurol. 56: 25-7, 1999. Ajchenbaum, F. et al. Independent regulation of human D-type cyclin gene expression during G1 phase in primary human T lymphocytes. J. Biol. Chem. 268:4113—9, 1993. Allen LA. The role of the neutrophil and phagocytosis in infection caused by Helicobacter pylori. Curr. Opin. Infect. Dis. 14: 273-7, 2001. Antal P, sipka S, Suranyi P, Csipo I, Seres T, Marodi L and Szegedi G. Flow cytometric assay of phagocytic activity of human neutrophils and monocytes in whole blood by neutral red uptake. Ann Hematol. 70: 259-65, 1995. Appleman, L.J. et al. CD28 costimulation mediates T cell expansion via IL-2-independent and IL-2-dependent regulation of cell cycle progression. J. Immunol. 164 : 144—51, 2000. Ashmore LM, Shopp GM and Edwards BS. Lymphocytesubset analysis by flow cytometry. Comparison of three different staining techniques and effects of blood storage. J. Immunol. Meth. 118: 209-15, 1989. Baehner RL and Nathan DG. Quantitative nitroblue tetrazolium test in chronic granulomatous disease. N. Engl. J. Med. 278: 971-6, 1968. Balomenos, D. et al. The cell cycle inhibitor p21 controls T-cell proliferation and sex-linked lupus development. Nat. Med. 6: 171—6, 2000. Barber GN. Host defense, viruses and apoptosis. Cell death Differ. 8: 113-24, 2001. Bambauer R and Arnold A. Plasmapheresis with a substitution solution of human serum protein (5%) versus plasmapheresis with a substitution solution of human albumin (5%) in patients suffering from autoimmune disease. Artificial Organs. 23: 1079-87, 1999. Beeson PB. Age and sex association of 40 autoimmune disease. Am. J. Med. 96: 457-62, 1994. Benoist C and D Mathis. Autoimmunity: The pathogen connection. Nature. 394: 227, 1994. Benget F, Sten O and Boris Z. Apoptosis in human disease: a new skin for the old ceremony? Biochemical and Biophysical Research Communication. 266: 699, 1999. Bernatz PE, Khonsari S and Harrison EG Jr. Thymoma: factors influencing prognosis. Surg Clin North Am , 53: 885-92, 1973. Boussiotis, V.A. et al. p27kip1 functions as an anergy factor inhibiting interleukin 2 transcription and clonal expansion of alloreactive human and mouse helper T lymphocytes. Nat. Med. 6, 290—297,.2000. Bromelow KV, Galea-Lauri J, O’Brien ME and Souberbielle BE. A highly sensitive whole blood natural killer cell assay. J. Immunol. Meth. 217: 177-84, 1998. Buckley C, Newsom-Davis J, Willcox N and Vincent A. Do titin and cytokine antibodies in MG patients predict thymoma or thymoma recurrence? Neurology. 57: 1579-82, 2001. Bukowski JF and Welsh RM. Inability of interferon to protect virus-infected cells against lysis by natural killer (NK) cells correlates with NK cell-mediated antiviral effects in vivo. J. Immunol. 135: 3537-41, 1985. Butcher EC and Picker LJ. Lymphocyte homong and homeostasis. Science. 272: 60-6, 1996. Cantinieaux B, Hariga C, Courtoy P, Hupin J and Fondu P. Staphylococc- us aureus phagocytosis: A new cytofluorometric method using FITC and paraformaldehyde. J. Immunol. Meth. 121: 203-8, 1989. Caroline CW. Sex differences in autoimmune disease. Nature. 2(9): 777-80, 2001. Chang L, Gusewitch GA, and Chritton DBW. Rapid flow cytometric assay for the assessment of natural killer cell activity. J. Immunol. Meth. 166: 45-54, 1993. Chaplin DD. The immune system: Overview of the immune response. J. Allergy Clin. Immunol.111: S442-59, 2003. Christensen PB, Jensen TS, Tsiropoulos I, Sorensen T, Kjaer M, Hojer-Pedersen E, Rasmussen MJ, and Lehfeldt E. Incidence and prevealence of myasthenia gravis in weater Denmark: 1975 to 1989. Neurology. 43: 1779-83, 1993. Chiu HC, Hsieh KH and Hung TP. Imbalances in T-cell subpopulations in myasthenia gravis. J. Neurol. Sci. 52: 53-9, 1981. Chiu HC, Hsieh KH and Hung TP. Effect of myasthenia serum and anti-acetylcholine receptor monoclonal antibody on the distribution of T-cell subsets in myasthenia patients and normal subjects. Asian. Pac. J. Allergy Immunol . 3: 30-6, 1985. Chiu HC, Hsieh RP and Hsieh KH. Association of HLA antigens with myasthenia gravis in Chinese on Taiwan. 23: 12-8, 1990. Concouvanis E and Martin GR. Siganl for death and survival: a two-step mechanism for cavitation in the vertebrate embryo. Cell 83: 279-87, 1995. Cohen MS. Epidemiology of myasthenia gravis. Monogr Allorgy. 21: 246-51, 1987. Cregan SP, Brown DL and Mitchel RE. Apoptosis and the adaptive response in human lymphocyte. Internat. J. Radia. Biol. 75: 1087-94, 1999. Dao T, Ohashi K, Kayano T, Kurimoto M and Okamura H. Interferon-γ-inducing factor, a novel cytokine, enhances Fas ligand-mediated cytotoxicity of murine T helper 1 cells. Cell. Immunol.. 173: 230-35, 1996. Dao T, Mehal WZ and Crispe IN. IL-18 augment perforin-dependent cytotoxicity of liver NK-T cells. J. Immunol. 161: 2217-22, 1998. David PR and Mark AA. Treatment of autoimmune myasthenia gravis. Neurol.. 61: 1652-61, 2003. Dunn PA and Tyrer HW. Quantitation of neutrophil phagocytosis, using fluorescent latex beads. J. Lab. Clin. Med. 98: 374-81, 1981. Elmqvist D, Hofmann W, Kugelberg J and Quastel D. An electrophysiological investigation of neuromuscular transmission in myasthenia gravis. J. Physiol. (Lond.) 174: 417-34, 1964. Erickson, S. et al. Involvement of the Ink4 proteins p16 and p15 in T-lymphocyte senescence. Oncogene 17: 595—602, 1998. Evoli A. Clinical heterogeneity of seronegative myasthenia gravis. Neuromuscul. Disord. 6: 155-61, 1996. Fadeel B, Orrenius S and Zhivotovsky B. Apoptosis in Human Disease: A New Skin for the Old Ceremony? Biochem. Biophysic. Res. Communi. 266: 699-717, 1999. Fishelson Z, Attali G and Mevorach D. Complement and apoptosis. Mol. Immunol. 38: 207-19, 2001. Fleisher TA. Apoptosis. Ann. Allergy Asthma Immunol. 78: 245-50, 1997. Franklin, D.S. et al. CDK inhibitors p18(INK4c) and p27(Kip1) mediate two separate pathways to collaboratively suppress pituitary tumorigenesis. Genes Dev. 15: 2899—911, 1998. Gennaro S, Fehder W and Gallagher P. Lymphocyte, monocyte, and natural killer cell reference ranges in postpartal women. Clin. Diagn. Lab. Immunol. 4: 195-201, 1997. Gema FF, María AR, Maria CG-P, Angel LM, Marcos LH and Manuel A. B lymphopenia in uraemia is related to an accelerated in vitro apoptosis and dysregulation of Bcl-2. Nephrol. Dial. Transplant. 15: 502-10, 2000. Godoy-Ramirez K, Franck K and Gaines H. A novel method for the simultaneous assessment of natural killer cell conjugate formation and cytotoxicity at the single-cell level by multi-parameter flow cytometry. J. Immunol. Meth. 239: 35-44, 2000. Goldsby RA, Osborne BA and Kindt TJ. Kuby Immunology. 4th edition. Chapter 1: Overview of the immune system. W.H. Freeman and Company. New York. pp.11-39, 2000. Gowans JL and Kinight EJ. The route of recirculation of lymphocytes in the rat. Proc. R. Soc.London Ser.B. 159: 257, 1964. Grattan CE, Dawn G, Gibbs S and Francis DM. Blood basophil numbers in chronic ordinary urticaria and healthy controls: diurnal variation, influence of loratadine and prednisolone and relationship to disease activity. Clin Exp Allergy. 33: 337-41, 2003. Griffith TS and Lynch DH. TRAIL: a molecule with multiple receptor and control mechanisms. Curr. Opin. Immunol. 10: 559-63, 1998. Harris EK. Statistical aspects of reference values in clinical pathology. Prog. Clin. Pathol. 8: 45-66, 1983. Hatcher FM and Kuhn RE. Destruction of Trypanosoma cruzi by natural killer cells. Science 218: 295-6, 1982. Hayashi T and Faustman DL. Implications of altered apoptosis in diabetes mellitus and autoimmune disease. Apoptosis 6: 31-45, 2001. Hedley DW, Shankey TV and Wheeless LL. DNA cytometry conseneue conference. Cytometry. 14: 471, 1993. Hofstra CL, Van AI, Hofman G, Kool M, Nijkamp FP and Van Oosterhout A. Prevention of Th2-like cell responses by coadministration of IL-12 and IL-18 is associated with inhibition of antigen-induced airway hyperresponsiveness, eosinophilia, and serum IgE levels. J. Immunol. 161: 5054-60, 1998. Hoshino K, Tsutsui H, Kawai T, Takeda K, Nakanishi K, Takeda Y and Akira S. Generation of IL-18 receptor-deficient mice: evidence for IL-1 receptor-related protein as an essential IL-18 binding receptor. J. Immunol. 162: 5041-44,1995 Howie, SEM, Harrison DJ and Wyllie AH. Lymphocyte apoptosis- mechanisms and implications in disease. Immunol. Rev. 142: 141-56, 1994. Hyodo Y, Matsui K, Hayashi N, Tsutsi H, Kashiwamura S, Yamauchi H, Hiroishi K, Takeda K, Tagawa Y, Iwakura Y, Kayagaki N, Kurimoto M, Okamura H, Hada T, Yagita H, Akira S, Nakanishi K and Higashino K. IL-18 upregulates perforin-mediated NK activity without increasing perforin messenger RNA expression by binding to constitutively expressed IL-18 receptor. J. Immunol. 162: 1662-68, 1999. Janeway CA. Natural killer cells: a primitive immune system. Nature 341: 108, 1989. Jana-Maria SM, Roland EW, Michael T, Waltraud M and Wolfgang R. Changes in lymphocytic cluster distribution during extracorporeal immunoadsorption. Artitif Organs. 26: 140-4, 2002. Janos GK, Marius P and Peter JS. Immunological effects of therapeutic immunoadsorption with respect to biocompatibility. Transfus. Sci. 19: 9-23, 1998. John HR and Timothy JL. Lymphocyte-mediated cytotoxicity. Annu. Rev. Immunol.. 20: 323-70, 2002. Kägi D, Ledermann B, Bürki K, Zinkernagel RM and Hengartner H. Molecular mechanisms of lymphocyte-mediated cytotoxicity and their role in immunological protection and pathogenesis in vivo. Annu. Rev. Immunol. 14: 207-32, 1996. Kadar JG, Spaeth PJ, Gaczkowski A, Oette K and Borberg H. Biocompatibility studies on a clinically well tolerated extracoreal system. Plasma. Ther. Transf. Technol 8: 307—18, 1987. Kaplan M.H. Stat proteins control lymphocyte proliferation by regulating p27kip1 expression. Mol. Cell. Biol. 18: 1996—2003, 1998. Kayagaki N, Yamaguchi N, Nakayama M, Takeda K, Akiba H, Tsutsui T, Okamura H, Nakanishi K, Okumura K and Yagita H. Expression and function of TNF-related apoptosis-inducing ligand(TRAIL) on murine activated NK cells. J. Immunol. 163: 1906-13, 1999. Kerr JFR, Wyllie AH and Currie AR. Apoptosis: a basic biological phenomenon with wide ranging implications in tissue kinetics. Br. J. Cancer. 26(4): 239-57, 1972. Keller AJ and Urbaniak SJ. Intensive plasma exchange on the cell separator. Effects on serum immunoglobulins and complement components. Br. J. Hematol. 531-4, 1978. Kiecolt-Glaser JK, Garner W, Speicher C, Penn GM, Holliday J and Glaser R. Psychosocial modifiers of immunocompetence in medical students. Psychosom. Med. 46: 7-14, 1984. Kiecolt-Glaser JK. Norman cousins memorial lecture 1998. Stress, personal relationships, and immune function: health implications. Brain, Behav. Immun. 13: 61-72, 1999. Klimas NG, Salvato FR, Morgan R and Fletcher MA. Immunologic abnormalities in chronic fatigue syndrome. J. Clin. Microbiol. 28: 1403-10, 1990. Konjevic G, Jurisic V, Banicevic B and Spuzic I. The difference in NK-cell activity between patients with non-Hodgkin’s lymphomas and Hodgkin’s disease. Br. J. Haematol. 104: 144-51, 1999. Kothakota S, Azuma T, Reinhard C and Klippel A. Caspase-3 generated fragment of gelsolin: effector of morphological change in apoptosis. Science 278: 294-8, 1997. Kott E, Hahn T, Huberman M, Levin S and Schattner. A Interferon system and natural killer cell activity in myasthenia gravis. Q J Med. S76:951-60, 1990. Kurosaka K, Watanabe N and Kobayashi Y. Production of Proinflammatory Cytokines by Resident Tissue Macrophages after Phagocytosis of Apoptotic Cells. Cell Immunol. 211: 1-7, 2001. Kwon, T.K. et al. The regulation of p27Kip1 expression following the polyclonal activation of murine G0 T cells. J. Immunol. 158 : 5642—8, 1997. Lam, E.W. et al. Cyclin D3 compensates for loss of cyclin D2 in mouse B lymphocytes activated via the antigen receptor and CD40. J. Biol. Chem. 275: 3479—84, 2000 Lape’-Nixon ML and Prince HE. How many gated lymphocytes are needed for accurate assessment of T-subset percentages by flow cytometry? Cytometry. 26: 223-6, 1996. Lee J and Desiderio S. Cyclin A/CDK2 regulates V(D)J recombination by coordinating RAG-2 accumulation and DNA repair. Immunity 11: 771—81, 1999. Lemke H, Schaefer R and Heidland A. Hypersensitivity reactions during hemodialysis: role of complement fragment and ethylene oxid antibodies. Nephrol. Dial. Transplant. 5: 264—9, 1990. Levine GD, Rosai J. Thymic hyperplasia and neoplasia : a review of current concepts. Hum Pathol. 9: 495-515, 1978. Lindstrom JM, Seybold ME, Lennon VA, Whittingham S and Duane DD. Antibody to acetylcholine receptor in myasthenia gravis. Prevalence,clinical correlates and diagnostic value. Neurology. 26: 1054-9, 1976. Lin JC. Myasthenia gravis and thymoma. Acta Neurol Taiwan. 12: 11-5, 2003. Lin, SC, Chou CC and Tsai MJ. Age-related changes in blood lymphocyte subsets of Chinese children. Pediatr. Allergy Immunol. 9: 215-20, 1998. Lozzio CB and Lozzio BB. Human chronic myelogenous leukemia cell-line with positive Philadelphia chromosome. Blood 45: 321-34, 1975. Marie-Lise G. Apoptosis as an HIV strategy to escape immune attack. Nature. 3: 392-404, 2003. Massoka A, Monden Y, Nakahara K. Follow-up study of thymamas with special reference to their clinical stages. Cancer. 407: 119-49, 1981. McCloskey TW, Oyaizu N, Coronesi M, and Pahwa S. Use of a flow cytometric assay to quantitate apoptosis in human lymphocytes. Clin. Immunol. Immunopathol. 71: 14-8, 1994. Mellqvist UH, Hansson M, Brune M, Dahlgren C, Hermodsson S and Hellstrand K. Natural killer cell dysfunction and apoptosis induced by chronic myelogenous leukemia cells: role of reactive oxygen species and regulation by histamine. Blood 96: 1961-8, 2000. Merad M, Manz MG, Karsunky H, Wagers A, Peters W, Charo I, Weissman IL, Cyster JG and Engleman EG. Langerhans cells renew in the skin throughout life under steady-state conditions. Nat. Immunol. 3: 1135-41, 2002. Miller RA. The aging immune system: primer and prospectus. Science. 273: 70-4, 1996. Morgan BP. Complement membrane attack on nucleated cells: resistance, recovery and non-lethal effects. Biochem. J. 264: 1—14, 1989. Morse, L. et al. Induction of cell cycle arrest and B cell terminal differentiation by CDK inhibitor p18 (INK4c) and IL-6. Immunity 6: 47—56, 1997. Mossman S, Vincent A and Newsom-Davis J. Myasthenia gravis without acetylcholine-receptor antibody: a distinct disease entity. Lancet 1: 116-9, 1986. Muller-Hermelink, H.K, Marx A. Thymoma. Curr. Opin. Oncol. 12: 426-33, 2000. Mullins, M.W. et al. CD40-mediated induction of p21 accumulation in resting and cycling B cells. Mol. Immunol. 35: 567—80, 1998. Murasko DM, Nelson BJ, Silver R, Matour D and aye D. Immunologic response in an elderly population with a mean age of 85. Am. J. Med. 81: 612-8, 1986. Mygland A. Ryanodine receptor autoantibodies in myasthenia gravis patients with a thymoma.. Ann. Neurol. 32: 589-91, 1992. Nagata S and Golstein P. The Fas death factor. Science. 267: 1449-56, 1995. Nagata S. Apoptosis by death factor. Cell. 88: 355-65, 1997. NCCLS H-42A. Clinical applications of flow cytometry: Quality assurance and immunophenotyping of lymphocytes; approved guideline. NCCLS Vol. 18: pp47. Neves MF, Starke-Buzetti. WA and Castro AMMG. Mast cell and eosinophils in the wall of the gut and eosinophils in the blood stream during Toxocara vitulorum infection of the water buffalo calves (Bubalus bubalis). Vet Parasitol. 113: 59-72, 2003. Nourse, J. et al. Interleukin-2-mediated elimination of the p27Kip1 cyclin-dependent kinase inhibitor prevented by rapamycin. Nature 372: 570—3, 1994. Oben JA and Foreman JC. A simple quantitative fluorimetric assay of in vitro phagocytosis in human neutrophils. J. immunol. Meth. 112: 99-103, 1988. Ojo-Amaize EA, Conley EJ and Peter JB. Decreased natural killer cell activity is associated with severity of chronic fatigue immune dysfunction syndrome. Clin. Infect. Dis. 18(Suppl 1): S157-9, 1994. Okamoto I, Kohno K, Tanimoto T, Ikegami H and Kurimoto M. Development of CD8+ effector T cell is differentially regulated by IL-18 and IL-12. J. Immunol. 162: 3202-11, 1999. Okamure H, Tsutsui H, Komatsu T, Yutsudo M, Hakura A, Tanimoto T, Torigoe K, Okura T, Nukada Y and Hattori K. Cloning of a new cytokine that induces IFN-γ production by T cells. Nature. 378: 88-91, 1995. Oosterhuis HJGH, Limburg PC, Hummel-Tappel E and The TH. Anti-acetylcholine receptor antibody in myasthenia gravis. Ⅱ. Clinical and serological follow-up of individual patients. J. Neurol. Sci. 58: 371-85, 1983. Osborne BA. Apoptosis and the maintenance of homoeostasis in the immune system. Curr. Opin. Immunol. 8: 245-54, 1996. Patki AH, Zielske SP, Sieg SF and Lederman MM. Preferential S phase entry and apoptosis of CD4+ T lymphocytes of HIV-1-infected patients after in vitro cultivation. Clin. Immunol. 97: 241-7, 2000. Picker LJ and Butcher EC. Physiological and molecular mechanism of lymphocyte homing. Annu. Rev. Immunol.. 10: 561-91, 1992. Perticarari S, Presani G and Banfi E. A new flow cytometric assay for the evaluation of phagocytosis and the oxidative burst in whole blood. J. Immunol. Meth. 170: 117-24, 1994. Perussia B, Tutt MM, Qiu WQ, Kuziel WA, Tucker PW, Trinchieri G, Bennett M, Ravetch JV and Kumar V. Murine natural killer cells express functional Fc gamma receptor II encoded by the Fc gamma R alpha gene. J. Exp. Med. 170: 73-86, 1989. Perussia B, Tutt MM, Qiu WQ, Kuziel WA, Tucker PW, Trinchieri G, Bennett M, Ravetch JV and Kumar V. Murine natural killer cells express functional Fc gamma receptor II encoded by the Fc gamma R alpha gene. J. Exp. Med. 170: 73-86, 1989. Poulas K, Tsibri E, Kokla A, Papanastasiou D, Tsouloufis T, Marinou M, Tsantili P, Papapetropoulos T and Tzartos SJ. Epidemiology of serpositive myasthenia gravis in Greece. J. Neurol. Neurosurg. 71: 352-6, 2001. Quintanilla-Martinez L, Wilkin EW and Ferry JA. Thymoma-morphologic subclassification correlates with invasiveness and immunohistologic features: a study of 122 cases. Hum Pathol. 24: 958-69, 1993 Ravagnan L, Roumier T and Kroemer G. Mitochondria, the killer organelles and their weapons. J. Cellular Physiol. 192: 131-137, 2002. Rosai J. Histological typing of tumours of the thymus. New York, NY: Spring-Verlag, 1999. Rudel T and Bokoch GM. Membrane and morphological changes in apoptotic cells regulated by caspase-mediated activation of PAK2. Science 276: 1571-1574, 1997. Sabzevari H. et al. G1 arrest and high expression of cyclin kinase and apoptosis inhibitors in accumulated activated/memory phenotype CD41 cells of older lupus mice. Eur. J. Immunol. 27: 1901—10, 1997. Salakou S, Tsamandas AC, Bonikos DS, Papapetropoulos T and Dougenis D. The potential role of bcl-2, bax, and Ki67 expression in thymus of patients with myasthenia gravis, and their correlation with clinicopathologic parameters. Eur. J. Cardio-Thoracic Surgery. 20: 712—21, 2001. Salama A, Hugo F and Heinrich D. Deposition of terminal C5b-9 complement complexes on erythrocytes and leukocytes during cardiopulmonary bypass. N. Engl. J. Med. 318: 408—14, 1988. Sakahira H, Enari M and Nagata S. Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature 391: 96-99, 1998. Scaffidi C, Kirchhoff S, Krammer PH and Peter ME. Apoptosis signaling in lymphocytes. Curr. Opin. Immunol. 11: 277-85, 1999. Schantz SP, Brown BW, Lira E, Taylor DL and Beddingfield N. Evidence for the role of natural immunity in the control of metastatic spread of head and neck cancer. Cancer immunol. Immunother. 25: 141-5, 1987. Schantz SP, Schillitoe EJ, Brown B and Campbell B. Natural killer cell activity and head and neck cancer: a clinical assessment. J. Natl. Cancer Inst. 77: 869-75, 1986. Shankey TV, Rabinovitch PS, Bagwell B, Bauer KD, Duque RE, Hedley DW, Mayall BH and Wheeless LL. Guidelines for implementation of clinical DNA cytometry. Cytometry. 14: 472-7, 1993. Sherr, C.J. Cancer cell cycles. Science 274: 1671—6, 1996. Sherr, C.J. and Roberts, J.M. CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev. 13: 1501—12, 1999. Smith ML and Fornace AJ. Mammalian DNA damage-inducible genes associated with growth arrest and apoptosis. Mutation Res. 340: 109-24, 1996. Sneller MC, Wang J, Dale JK, Strober W, Middelton LA, Choi Y, Fleisher TA, Lim MS, Jaffe ES, Puck JM, Lenardo MJ and Straus SE. Clinical, immunologic, and genetic features of an autoimmune lymphoproliferative syndrome associated with abnormal lymphocyte apoptosis. Blood 89: 1341-8, 1997. Son K, Kew M and Rabson AR. Depressed natural killer cell activity in patients with hepatocellular carcinoma. Cancer 50: 2820-5, 1982. Sprent J. T lymphocytes and the thymus. In fundamental Immunology, ed. WE Paul, pp.75-110. New York: Raven. 3ed ed, 1993. Sprent J. Recirculating lymphocytes. In the lymphocytes: Structure and function, ed. JJ Marchalonis, pp.43-112. New York: Dekker, 1997. Stear MJ, Henderson NG, Kerr A, McKellar QA, Mitchell S, Seeley C and Bishop SC. Eosinophilia as a marker of resistance to Teladorsagia circumcincta in Scottish Blackface lambs. Parasitology. 124: 553-60, 2002. Strayer DR, Carter WA and Brodsky I. Familial occurrence of breast cancer is associated with reduced natural killer cytotoxicity. Breast Cancer Res. Treat. 7: 187-92, 1986. Tamir, A. and Miller, R.A. Aging impairs induction of cyclindependent kinases down-regulation of p27 in mouse CD41 cells. Cell Immunol. 198: 11—20, 1999. Theofilopulos AN. The basis of autoimmunity. Part Ⅰ: Mechanism of aberrant self-recognition. Immunol. Today. 16: 90, 1995. Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science 267: 1456-62, 1995. Trinchieri G. Interleukin-12; a cytokine at the interface of inflammation and immunity. Adv. Immunol. 70: 83-243, 1998. Trinchieri G and Perussia B. Human natural killer cells: biologic and pathologic aspects. Lab. Invest. 50: 489-513, 1984 Trinchieri G. Biology of natural killers. Adv. Immunol. 47: 187-376, 1989. Tsutsui H, Nakanishi K, Matsui K, Higashino H, Okamura Y and Miyszawa Kaneda K. Interferon-γ-inducing factor up-regulates Fas ligand-mediated cytotoxic activity of murine natural killer cell clones. J. Immunol. 157:3967-73, 1996. Ushio S, Namba M, Okura T, Hattori K, Nukada Y, Akita K, Tanabe F, Konishi K, Micallef M, Fujii M, Torigoe K, Tanimoto T, Fukuda S, Ikeda M, Okamura H and Kurimoto M. Cloning of the cDNA for human IFN-γ-inducing factor, expression in Escherichia coli, and studies on the biologic activities of the protein. J. Immunol. 156: 4274-79, 1996. Van Eeden SF, Klut ME and Walker BAM. The use of flow cytometry to measure neutrophil function. J. Immunol. Meth. 232: 23-43, 1999. Vizler C, Nagy T, Kusz E, Glavinas H and Duda E. Flow cytometric cytotoxicity assay for measuring mammalian and avian NK cell activity. Cytometry. 47: 158-62, 2002. Vojdani A, Ghoneum M and Choppa P. Minimizing cancer risk using molecular techniques: a review. Toxicol. Ind. Health. 13: 589-626, 1997a. Vojdani A, Ghoneum M, Choppa PC, Magtoto L and Lapp CW. Elevated apoptotic cell population in patients with chronic fatigue syndrome: the pivotal role of protein kinase RNA. J. Intern. Med. 242: 465-78, 1997b. Wan CP, park CS and Lau BHS. A rapid and simple microfluorometric phagocytosis assay. J. Immunol. Meth. 162: 1-7, 1993. Wenisch C, Patruta S and Daxbock F. Effect of age on human neutrophil function. J. Leukoc. Biol. 67: 40-5, 2000. White-Owen C, Alexander JW and Sramkoski RM. Rapid whole-blood microassay using flow cytometry for measuring neutrophil phagocytosis. J. Clin. Microbiol. 30: 2071-6, 1992. Whiteside TL and Friberg D. Natural killer cells and natural killer cell activity in chronic fatigue syndrome. Am. J. Med. 105: 27S-34S, 1998. Whiteside TL and Herberman RB. Short analytical review. The role of natural killer cells in human disease. Clin. Immunol. Immunopathol. 53: 1-23, 1989. Whiteside TL and Herberman RB. The role of natural killer cells in immune surveillance of cancer. Curr. Opin. Immunol. 7: 704-10, 1995. Wiley SR, Schooley K, Smolak PJ, Din WS, Huand C-P, Nicholl JK, Sutherland GR, Smith TD, Rauch C, Smith CA and Goodwin RG. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity. 3: 673-82, 1995. Wyllie AH. Apoptosis and the regulation of cell numbers in normal and neoplastic tissues: an overview. Cancer Metastasis Rev. 11: 95-103, 1992. Xaus, J. et al. Interferon g induces the expression of p21waf-1 and arrests macrophage cell cycle, preventing induction of apoptosis. Immunity 11: 103—13, 1999. Yeh JH and Chiu HC. Double filtration plasmapheresis in myasthenis gravis-analysis of clinical efficacy and prognostic parameters. Acta Neurol Scand. 100: 305-9, 1999. Yeh JH, Chen WH and Chiu HC. Predicting the course of myasthenic weakness following double filtration plasmapheresis. Acta Neurol Scand. 108: 174-8, 2003. Yoshimoto T, Okamura H, Tagawa YI, Iwakura Y and Nakanishi K. Interleukin 18 together with interleukin 12 inhibits IgE production by induction of interferon-γ production from activated B cells. Proc. Natl. Acad. Sci. 94:3948-53, 1997. Yoshimoto T, Tsutsui H, Tominaga K, Hoshino K, Okamura H, Akira S, Paul WE and Nakanishi K. Interleukin 18, although antiallergic when administered with IL-12, stimulates IL-4 and histamine release by basophils. Proc. Natl. Acad. Sci. 96:13962-66, 1999. Yuan J and Yankner BA. Apoptosis in the nervous system. Nature. 407: 802-9, 2000. Zhang, P. et al. The cell cycle and development: redundant roles of cell cycle regulator. Curr. Opin. Cell Biol. 11: 655—62, 1999. 邱浩彰,謝健明,陳榮基,林芳郁。中國人肌無力症之胸腺切除。中華微免雜誌 20: 15-22, 1987.

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系統識別號 U0007-1704200715053623
論文名稱(中文) 人類乙醯轉移?基因型與腦瘤之關聯研究
論文名稱(英文) The Correlation between Human N-acetyltransferase 2 Genotypes and Brain Tumors
校院名稱 臺北醫學大學
系所名稱(中) 藥學研究所
系所名稱(英) Graduate Institute of Pharmacy
學年度 94
學期 2
出版年 95
研究生(中文) 蕭珮妤
學號 M301093014
學位類別 碩士
語文別 中文
口試日期
論文頁數 71頁
口試委員 指導教授-李仁愛
指導教授-陳香吟
關鍵字(中) 乙醯轉移?
腦瘤
代謝多型性
基因型
危險因子
人類
病患組
對照組
臺灣
關鍵字(英) N-acetyltransferase
Brain Neoplasms
Metabolic polymorphism
Genotype
Risk factors
Human
Taiwan
學科別分類
中文摘要 腦瘤佔臺灣地區所有癌症死亡原因之1.25%,其五年存活率僅約20%。目前有關腦瘤的成因仍未明,已確認的危險因子僅有游離輻射一項。人類第二型乙醯轉移?(NAT2)參與許多致癌物的活化或解毒過程,其代謝速率有顯著的個體差異,一般以帶有一個以上野生型對偶基因(NAT2*4)者為rapid acetylator,其餘基因型稱為slow acetylator。NAT2和許多癌症有關,但和台灣地區腦瘤的相關則尚未有研究。 本研究主要目的為探討乙醯轉移?基因型的分布在腦瘤病患與未罹患癌症之配對對照組中的差異。次要目的為分析不同乙醯轉移?基因型的腦瘤病患其存活率、腫瘤型態、腫瘤分級之差異。 本研究比較27名腦瘤病患及27名正常對照組,以PCR-RFLP方式分析病患之腫瘤切片及對照組的週邊血液檢體,檢測NAT2*5、NAT2*6及NAT2*7,共三個allele。結果發現NAT2*7 allele在腦瘤病患組的比例明顯較對照組高,p=0.001,帶有NAT2*7 allele者罹患腦瘤之odds ratio為6.79(95%, CI 2.06-22.37);帶有NAT2*7 allele的病患中astrocytoma和glioblastoma multiforme的比例較高(p=0.016)。其他次要目的如存活、率腫瘤類型等則未發現有差異。因此,NAT2*7 allele與astrocytoma和glioblastoma multiforme之間的關聯值得進一步探討研究。
英文摘要 Brain tumors accounts for 1.25% of cancer-related death in Taiwan, and the 5-year survival is only 20%. The cause of brain tumors was not fully understood, and the only known risk factor is ionizing radiation. Previous studies have found that the highly polymorphic arylamine N-acetyltransferase 2 (NAT2) is related to many cancer development probably due to both activation and inactivation reaction of numerous carcinogens. Subjects with wild type allele NAT2*4 were called rapid acetylators, and others were called slow acetylators. The correlation of NAT2 and brain tumors in Taiwan has not yet been studied. The primary purpose of this study was to compare the distribution of acetylator types between brain tumor patients and age-matched normal subjects in Taiwan. The secondary purpose was to compare the progression-free survival, overall survival, and tumor type and grading of brain tumor patients with different acetylator types. We studied the NAT2* polymorphisms (NAT2*5, NAT2*6, NAT2*7) in 27 brain tumor tissues and 27 matched control samples from peripheral blood by the PCR-RFLP method. The allele frequency of NAT2*7 was found significantly higher in case group, p=0.001 (odds ratio 6.79, 95% CI, 2.06-22.37). The most common types of tumors for the patients with NAT2*7 were astrocytoma and glioblastoma multiforme; while those for the patients without NAT2*7 were oligodendroglioma, meningioma, and glioblastoma multiforme. No differences were found in survival or tumor grading. Further studies are warranted to clarify the relationship between NAT2*7 and occurance of astrocytoma and glioblastoma multiforme.
論文目次 第1章 前言 第2章 文獻探討 2.1 腦瘤概述 2.1.1 腦瘤簡介 2.1.2 腦瘤治療現況 2.1.3 腦瘤致病危險因子 2.2 代謝?與癌症 2.2.1 致癌物質的代謝 2.2.2 代謝?個體差異與癌症罹病率的關係 2.3 乙型乙醯轉移?(ARYLAMINE N-ACETYLTRANSFERASE 2) 2.3.1 乙型乙醯轉移?(Arylamine N-Acetyltransferase 2)簡介 2.3.2 乙型乙醯轉移?的基因多型性 2.3.3 乙型乙醯轉移?與癌症的關係 第3章 研究方法 3.1 研究設計 3.2 基因型檢測方式 3.2.1 DNA萃取 3.2.2 套疊聚合?連鎖反應—限制?切割片段長度多型性 3.2.3 基因型判定 3.3 統計分析 第4章 研究結果 4.1 病患組與對照組之結果比較 4.2 不同乙醯化代謝類型病患之結果比較 4.3 NAT2*7對偶基因子群體(SUBGROUP)之結果比較 第5章 討論 5.1 研究結果討論 5.2 研究限制及改進方向 第6章 結論 參考文獻
參考文獻 1.中華民國九十三年死因結果統計摘要. 2005. (Accessed 4/30, 2006, at http://www.doh.gov.tw/statistic/data/死因摘要/93年/93.htm.) 2.Wrensch M, Minn Y, Chew T, Bondy M, Berger MS. Epidemiology of primary brain tumors: current concepts and review of the literature. Neuro-oncol 2002;4(4):278-99. 3.Lin HJ, Han CY, Lin BK, Hardy S. Ethnic distribution of slow acetylator mutations in the polymorphic N-acetyltransferase (NAT2) gene. Pharmacogenetics 1994;4(3):125-34. 4.Hein DW. N-acetyltransferase 2 genetic polymorphism: effects of carcinogen and haplotype on urinary bladder cancer risk. Oncogene 2006;25(11):1649-58. 5.Kleihues P, Louis DN, Scheithauer BW, et al. The WHO classification of tumors of the nervous system. J Neuropathol Exp Neurol 2002;61(3):215-25; discussion 26-9. 6.Kleihues P, Cavenee WK. World Health Organisation classification of tumours: Pathology & Genetics of the Tumours of the Nervous System. Lyon: IARC Press; 2000. 7.Vandenberg SR, Lopes MBS. Classification. In: Berger MS, Wilson CB, eds. The Gliomas. 1st ed. Philadelphia: W. B. Saunders Company; 1999:172-91. 8.行政院衛生署國民健康局. 中華民國九十一年度癌症登記報告; 2005. 9.Ferlay J, Bray F, Pisani P, Parkin D. Globocan 2000: Cancer incidence, mortality and prevalence worldwide. Lyon: IARC Press; 2000. 10.Oi S, Matsuzawa K, Choi JU, Kim DS, Kang JK, Cho BK. Identical characteristics of the patient populations with pineal region tumors in Japan and in Korea and therapeutic modalities. Childs Nerv Syst 1998;14(1-2):36-40. 11.TCOG顱內腫瘤研究會. 腦瘤之診斷與治療共識: 國家衛生研究院 癌症研究組 臺灣癌症臨床研究合作組織; 2004. 12.Louis DN, Cavenee WK. Chapter 39--Neoplasms of the Central Nervous System. In: DeVita VT, Hellman S, Rosenberg SA, eds. Cancer--Principles & Practice of Oncology. 7th ed. Philadelphia: Lippincott Williams & Wilkins; 2005:1827-87. 13.Ohgaki H, Kleihues P. Epidemiology and etiology of gliomas. Acta Neuropathol (Berl) 2005;109(1):93-108. 14.Neglia JP, Meadows AT, Robison LL, et al. Second neoplasms after acute lymphoblastic leukemia in childhood. N Engl J Med 1991;325(19):1330-6. 15.Elexpuru-Camiruaga J, Buxton N, Kandula V, et al. Susceptibility to astrocytoma and meningioma: influence of allelism at glutathione S-transferase (GSTT1 and GSTM1) and cytochrome P-450 (CYP2D6) loci. Cancer Res 1995;55(19):4237-9. 16.Kelsey KT, Wrensch M, Zuo ZF, Miike R, Wiencke JK. A population-based case-control study of the CYP2D6 and GSTT1 polymorphisms and malignant brain tumors. Pharmacogenetics 1997;7(6):463-8. 17.Cancer: causes, occurrence and control. IARC Sci Publ 1990(100):1-352. 18.Luch A. Nature and nurture - lessons from chemical carcinogenesis. Nat Rev Cancer 2005;5(2):113-25. 19.Hirvonen A. Polymorphisms of xenobiotic-metabolizing enzymes and susceptibility to cancer. Environ Health Perspect 1999;107 Suppl 1:37-47. 20.Poirier MC, Santella RM, Weston A. Carcinogen macromolecular adducts and their measurement. Carcinogenesis 2000;21(3):353-9. 21.Guengerich FP. Metabolism of chemical carcinogens. Carcinogenesis 2000;21(3):345-51. 22.Kapitulnik J, Wislocki PG, Levin W, Yagi H, Jerina DM, Conney AH. Tumorigenicity studies with diol-epoxides of benzo(a)pyrene which indicate that (+/-)-trans-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene is an ultimate carcinogen in newborn mice. Cancer Res 1978;38(2):354-8. 23.Wislocki PG, Kapitulnik J, Levin W, Conney AH. Tumorigenicity of benzo[alpha]pyrene 4,5-,7,8-,9,10- and 11,12-oxides in newborn mice. Cancer Lett 1978;5(4):191-7. 24.Bourne D. A First Course in Pharmacokinetics and Biopharmaceutics. In: Flying Publisher; 2002. 25.Arlt VM, Glatt H, Muckel E, et al. Metabolic activation of the environmental contaminant 3-nitrobenzanthrone by human acetyltransferases and sulfotransferase. Carcinogenesis 2002;23(11):1937-45. 26.Chung FL, Chen HJ, Nath RG. Lipid peroxidation as a potential endogenous source for the formation of exocyclic DNA adducts. Carcinogenesis 1996;17(10):2105-11. 27.Borghoff SJ, Short BG, Swenberg JA. Biochemical mechanisms and pathobiology of alpha 2u-globulin nephropathy. Annu Rev Pharmacol Toxicol 1990;30:349-67. 28.Guengerich FP, Shimada T. Oxidation of toxic and carcinogenic chemicals by human cytochrome P-450 enzymes. Chem Res Toxicol 1991;4(4):391-407. 29.Rodriguez-Antona C, Ingelman-Sundberg M. Cytochrome P450 pharmacogenetics and cancer. Oncogene 2006;25(11):1679-91. 30.Raunio H, Husgafvel-Pursiainen K, Anttila S, Hietanen E, Hirvonen A, Pelkonen O. Diagnosis of polymorphisms in carcinogen-activating and inactivating enzymes and cancer susceptibility--a review. Gene 1995;159(1):113-21. 31.Ariyoshi N, Miyamoto M, Umetsu Y, et al. Genetic polymorphism of CYP2A6 gene and tobacco-induced lung cancer risk in male smokers. Cancer Epidemiol Biomarkers Prev 2002;11(9):890-4. 32.Ketterer B, Harris JM, Talaska G, et al. The human glutathione S-transferase supergene family, its polymorphism, and its effects on susceptibility to lung cancer. Environ Health Perspect 1992;98:87-94. 33.McIlwain CC, Townsend DM, Tew KD. Glutathione S-transferase polymorphisms: cancer incidence and therapy. Oncogene 2006;25(11):1639-48. 34.Lohmueller KE, Pearce CL, Pike M, Lander ES, Hirschhorn JN. Meta-analysis of genetic association studies supports a contribution of common variants to susceptibility to common disease. Nat Genet 2003;33(2):177-82. 35.Butcher NJ, Boukouvala S, Sim E, Minchin RF. Pharmacogenetics of the arylamine N-acetyltransferases. Pharmacogenomics J 2002;2(1):30-42. 36.Enzyme Nomenclature---EC 2.3.1.5. 1992. (Accessed 2 April, 2006, at http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/3/1/5.html.) 37.Hein DW, McQueen CA, Grant DM, Goodfellow GH, Kadlubar FF, Weber WW. Pharmacogenetics of the arylamine N-acetyltransferases: a symposium in honor of Wendell W. Weber. Drug Metab Dispos 2000;28(12):1425-32. 38.Grant DM, Blum M, Beer M, Meyer UA. Monomorphic and polymorphic human arylamine N-acetyltransferases: a comparison of liver isozymes and expressed products of two cloned genes. Mol Pharmacol 1991;39(2):184-91. 39.Grant DM, Morike K, Eichelbaum M, Meyer UA. Acetylation pharmacogenetics. The slow acetylator phenotype is caused by decreased or absent arylamine N-acetyltransferase in human liver. J Clin Invest 1990;85(3):968-72. 40.Meyer UA. Pharmacogenetics - five decades of therapeutic lessons from genetic diversity. Nat Rev Genet 2004;5(9):669-76. 41.Ebisawa T, Deguchi T. Structure and restriction fragment length polymorphism of genes for human liver arylamine N-acetyltransferases. Biochem Biophys Res Commun 1991;177(3):1252-7. 42.Sinclair JC, Sandy J, Delgoda R, Sim E, Noble ME. Structure of arylamine N-acetyltransferase reveals a catalytic triad. Nat Struct Biol 2000;7(7):560-4. 43.Jounela AJ, Pasanen M, Mattila MJ. Acetylator phenotype and the antihypertensive response to hydralazine. Acta Med Scand 1975;197(4):303-6. 44.Huang Y-S, Chern H-D, Su W-J, et al. Polymorphism of the N-acetyltransferase 2 gene as a susceptibility risk factor for antituberculosis drug-induced hepatitis. Hepatology 2002;35(4):883-9. 45.Vatsis KP, Weber WW, Bell DA, et al. Nomenclature for N-acetyltransferases. Pharmacogenetics 1995;5(1):1-17. 46.Matas N, Thygesen P, Stacey M, Risch A, Sim E. Mapping AAC1, AAC2 and AACP, the genes for arylamine N-acetyltransferases, carcinogen metabolising enzymes on human chromosome 8p22, a region frequently deleted in tumours. Cytogenet Cell Genet 1997;77(3-4):290-5. 47.Arylamine N-Acetyltransferase (NAT) Nomenclature. 2004. (Accessed 2 April, 2006, at http://www.louisville.edu/medschool/pharmacology/NAT.html.) 48.Fretland AJ, Leff MA, Doll MA, Hein DW. Functional characterization of human N-acetyltransferase 2 (NAT2) single nucleotide polymorphisms. Pharmacogenetics 2001;11(3):207-15. 49.Zhu Y, Doll MA, Hein DW. Functional genomics of C190T single nucleotide polymorphism in human N-acetyltransferase 2. Biol Chem 2002;383(6):983-7. 50.Cascorbi I, Brockmoller J, Bauer S, Reum T, Roots I. NAT2*12A (803A-->G) codes for rapid arylamine n-acetylation in humans. Pharmacogenetics 1996;6(3):257-9. 51.Hein DW, Doll MA, Rustan TD, Ferguson RJ. Metabolic activation of N-hydroxyarylamines and N-hydroxyarylamides by 16 recombinant human NAT2 allozymes: effects of 7 specific NAT2 nucleic acid substitutions. Cancer Res 1995;55(16):3531-6. 52.Ait Moussa L, Khassouani CE, Hue B, Jana M, Begaud B, Soulaymani R. Determination of the acetylator phenotype in Moroccan tuberculosis patients using isoniazid as metabolic probe. Int J Clin Pharmacol Ther 2002;40(12):548-53. 53.Jetter A, Kinzig-Schippers M, Illauer M, et al. Phenotyping of N- acetyltransferase type 2 by caffeine from uncontrolled dietary exposure. Eur J Clin Pharmacol 2004;60(1):17-21. 54.Hutchings A, Routledge PA. A simple method for determining acetylator phenotype using isoniazid. Br J Clin Pharmacol 1986;22(3):343-5. 55.Smith CA, Wadelius M, Gough AC, Harrison DJ, Wolf CR, Rane A. A simplified assay for the arylamine N-acetyltransferase 2 polymorphism validated by phenotyping with isoniazid. J Med Genet 1997;34(9):758-60. 56.Gross M, Kruisselbrink T, Anderson K, et al. Distribution and concordance of N-acetyltransferase genotype and phenotype in an American population. Cancer Epidemiol Biomarkers Prev 1999;8(8):683-92. 57.Deguchi T, Mashimo M, Suzuki T. Correlation between acetylator phenotypes and genotypes of polymorphic arylamine N-acetyltransferase in human liver. J Biol Chem 1990;265(22):12757-60. 58.Parkin DP, Vandenplas S, Botha FJ, et al. Trimodality of isoniazid elimination: phenotype and genotype in patients with tuberculosis. Am J Respir Crit Care Med 1997;155(5):1717-22. 59.Kukongviriyapan V, Prawan A, Tassaneyakul W, Aiemsa-Ard J, Warasiha B. Arylamine N-acetyltransferase-2 genotypes in the Thai population. Br J Clin Pharmacol 2003;55(3):278-81. 60.Hirvonen A. Polymorphic NATs and cancer predisposition. IARC Sci Publ 1999(148):251-70. 61.Chiou H-L, Wu M-F, Chien W-P, et al. NAT2 fast acetylator genotype is associated with an increased risk of lung cancer among never-smoking women in Taiwan. Cancer Lett 2005;223(1):93-101. 62.Trizna Z, de Andrade M, Kyritsis AP, et al. Genetic polymorphisms in glutathione S-transferase mu and theta, N-acetyltransferase, and CYP1A1 and risk of gliomas. Cancer Epidemiol Biomarkers Prev 1998;7(6):553-5. 63.Peters ES, Kelsey KT, Wiencke JK, et al. NAT2 and NQO1 polymorphisms are not associated with adult glioma. Cancer Epidemiol Biomarkers Prev 2001;10(2):151-2. 64.Bland JM, Altman DG. Survival probabilities (the Kaplan-Meier method). Bmj 1998;317(7172):1572. 65.Leon SP, Zhu J, Black PM. Genetic aberrations in human brain tumors. Neurosurgery 1994;34(4):708-22. 66.Ag?ndez J. NAT2 Genotyping: Equilibrium Between Accuracy and Feasibility in Routine Analyses. J Appl Res 2003;3(2):118-23.

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系統識別號 U0007-2107200815013300
論文名稱(中文) 粒線體DNA匱乏C6神經膠瘤細胞對於神經醯胺誘發細胞凋亡之抗性研究
論文名稱(英文) Resistance to Ceramide-induced Apoptosis in the Mitochondrial DNA-depleted C6 Glioma Cells
校院名稱 臺北醫學大學
系所名稱(中) 醫學科學研究所
系所名稱(英) Graduate Institute of Medical Sciences
學年度 96
學期 2
出版年 97
研究生(中文) 謝郁慧
學號 M102095006
學位類別 碩士
語文別 中文
口試日期 2008-07-04
論文頁數 66頁
口試委員 委員-魏耀揮教授
委員-謝秀梅副教授
委員-高淑慧助理教授
共同指導教授-謝榮鴻副教授
指導教授-郭泰志助理教授
關鍵字(中) mtDNA套數
溴化乙醯
二脫氧胞苷
細胞色素C
細胞凋亡
神經醯胺
關鍵字(英) mtDNA copy number
EtBr, ddC
Cytochrome c
apoptosismtDNA copy number
EtBr
ddC
Cytochrome c
apoptosis
ceramideceramide
學科別分類
中文摘要 粒線體在細胞凋亡發生的訊號傳遞途徑中扮演著一個重要調節者的角色。粒線體DNA (mtDNA)匱乏的細胞株對於TRAIL (TNF-related apoptosis-inducing ligand)所誘發的細胞凋亡感受性極低。在本實驗中我們利用溴化乙醯(ethidium bromide, EtBr)及二脫氧胞苷(zalcitabine, 2'-3'-dideoxycytidine, ddC)處理C6神經膠瘤細胞(C6 glioma cell)後,以同步定量聚合酶連鎖反應來偵測mtDNA套數,確認得到不同程度mtDNA匱乏的細胞,將所有藥劑移除72小時之後再次測量mtDNA套數的回復情形。各組細胞中mtDNA套數約為:control組, 100%; 1μM EtBr組, 52%; 25 μM EtBr組, 13%; 5 μM ddC組, 25%; re-1 μM EtBr組, 92%; re-25 μM EtBr組, 52%; re-5 μM ddC組, 51%。細胞的生長速率以25 μM EtBr組生長速率最低,細胞存活率以25 μM EtBr組細胞存活率最低。粒線體膜電位(mitochondrial membrane potential, △Ψ) 的測量結果顯示添加EtBr均導致△Ψ下降。實驗結果發現mtDNA套數的下降在本實驗中與粒線體ATP的形成能力無絕對關係。觀察粒線體電子傳遞鏈酵素群蛋白質(ND6、Core 2及F1α)的表現量後,結果顯示mtDNA套數的下降直接影響mtDNA調控的蛋白質ND6,但在藥劑移除之後表現量大幅高於控制組,顯示有抑制回饋現象。在確認了mtDNA套數的下降所造成的細胞生理的影響之後,使用共軛焦顯微鏡偵測細胞色素C (cyt c)的轉位作用及以流式細胞儀計算細胞凋亡的發生率。實驗結果顯示只有25 μM EtBr組具有明顯的cyt c轉位作用及較高的細胞凋亡發生率。若以25 μM ceramide誘發細胞凋亡,發現部分mtDNA套數下降的細胞對其細胞凋亡的誘發敏感性較小。而先使用3 μM cyclosporin A (CsA) 處理30 分鐘再以25 μM ceramide誘發細胞凋亡,各組細胞皆有細胞凋亡發生率下降的現象,顯示其細胞凋亡確實是由ceramide所誘發。實驗證實mtDNA套數確實會影響細胞生理功能,並且對於ceramide所誘發的細胞凋亡具有抗性。
英文摘要 Mitochondria is one of the master regulators in apoptosis signaling pathway. Mitochondrial DNA (mtDNA)-depleted cell line has lower sensitivity to apoptosis which inducing by TRAIL (TNF-related apoptosis-inducing ligand). In this study, we created C6 glioma cells with mtDNA depletion by treatment with ethidium bromide (EtBr) and zalcitabine (ddC) for 72 hours. The mtDNA copy numbers were determined from cells treated with EtBr and ddC followed removing incubated reagent 72 hours. MtDNA copy numbers were determined by with real-time polymerase chain reaction (real-time PCR) and listed as followed list: control: 100%, 1μM EtBr: 52%, 25 μM EtBr: 13%, 5 μM ddC: 25%, re-1 μM EtBr: 92%, re-25 μM EtBr: 52%, re- 5 μM ddC: 51%. We counted cell number for cell growth rate. The cell viabilities were also analyzed by MTS assay. Mitochondrial membrane potential (△Ψ) analyzed by JC-1 stainning indicated that EtBr groups had lowest △Ψ. Contents of cellular ATP generation were determined represented that ditacted lower mtDNA copy number was not direct proportion to ATP generation. Estimated mitochondrial electron transport chain (ETC) proteins (ND6, Core 2 and F1α) expression with Western blotting, the result indicated that cells harboring lower mtDNA copy number had lower mtDNA encoded protein, but mtDNA copy number restores and more higher than control cell after all drugs were removed. To confirme the influence of mtDNA depleting to cells, we observed the Cytochrome c (cyt c) translocation with confocol microscopy and calculated apoptotic proportion with flow cytometery. The result showed only the 25 μM EtBr treatment group has conspicuous effect of cyt c translocation and higher apoptotic ratio. The cell apoptosis induced with 25 μM ceramide showed that mtDNA-depleted cells were not susceptible for ceramide treatment. Moreover, cells incubated with 3 μM cyclosporine A (MPT pore inhibitor, CsA) for 30 min before 25 μM ceramide treatment showed lower apoptosis proportion occurred in all groups, this data indicated the cell apoptosis was induced by ceramide. According to our results, we suggest that decreased mtDNA copy number affected cell physiology and resisted to ceramide-inducing apoptosis.
論文目次 摘要 I
Abstract III
目錄 V
表目錄 IX
圖目錄 X
縮寫表 XII
藥品試劑列表 XIV
第一章 緒論 1
第二章 文獻回顧 2
第一節、 粒線體( Mitochondria ) 2
1.粒線體概述 2
2. mtDNA匱乏細胞株(ρ0 cell) 3
3.粒線體滲透性通透孔(Mitochondrial permeability transition pore, mPTP) 3
4. 粒線體與細胞死亡 5
第二節、 神經膠瘤細胞(Glioma cells) 6
第三節、 細胞凋亡( Apoptosis ) 6
1. 細胞凋亡的意義 7
2. 細胞凋亡的特徵 7
3. ρ0 cell與細胞凋亡 8
第四節、 神經醯胺(ceramide) 10
第三章 實驗材料與方法 11
第一節、藥品的配製 11
1. 溴化乙醯(EtBr)的配製 11
2. 二脫氧胞苷(Zalcitabine, 2'-3'-dideoxycytidine, ddC)的配製 12
3. 神經醯胺(Ceramide)的配製 12
4. 環孢靈(Cyclosporin A, CsA)的配製 12
第二節、實驗方法 13
一、 C6神經膠瘤細胞株( C6 glioma cell line )培養 13
二、細胞生長速率 13
三、細胞存活率(cell viability)測試 13
四、 DNA萃取 14
五、同步定量聚合酶連鎖反應(real-time polymerase chain reaction;real-time PCR) 14
六、蛋白質萃取及定量 15
七、西方墨點法分析(Western blot) 16
八、分析粒線體膜電位 17
九、細胞免疫染色 18
十、檢測ATP的產生 19
十一、 Annexin V-FITC 分析 20
十二、統計方法 20
第四章 實驗結果 21
第一節 神經膠瘤細胞生長分析 21
第二節 細胞存活率測試 22
第三節 神經膠瘤細胞mtDNA套數分析 22
第四節 神經膠瘤細胞粒線體膜電位(ΔΨ)分析 23
第五節 神經膠瘤細胞ATP變化情形 24
第六節 神經膠瘤細胞粒線體中蛋白質表現量改變情形 25
第七節 不同濃度ceramide處理之細胞存活率 26
第八節 神經膠瘤細胞經25 μM ceramide處理後其細胞色素C的轉位情形 27
第九節 神經膠瘤細胞經25 μM ceramide處理後其細胞凋亡情形 27
第五章 討論 30
第一節 mtDNA套數影響細胞生理功能 30
第二節 mtDNA套數影響細胞凋亡發生率 34
第六章 結論 37
第七章 參考文獻 38

表目錄
表 1、分析粒線體DNA基因之real-time PCR引子序列表及片段大小 45
表 2、一級抗體與二及抗體 46
表 3、實驗結果綜合比較表 47


圖目錄

圖 1、大鼠mtDNA圖 48
圖 2、電子傳遞鏈上Complex I~V的次單位分別由nuclar DNA (nDNA)與mtDNA轉錄/轉譯之蛋白質所組成。 49
圖 3、神經膠瘤細胞經EtBr或ddC處理後細胞生長倍數 52
圖 4、細胞存活率測試 53
圖 5、神經膠瘤細胞mtDNA套數分析 54
圖 6、神經膠瘤細胞粒線體膜電位 (△Ψ)變化情形 55
圖 7、神經膠瘤細胞ATP變化情形 56
圖 8、神經膠瘤細胞粒線體中蛋白質表現量改變情形 57
圖 9、不同濃度ceramide處理細胞後之細胞存活率 59
圖 10、以共軛焦顯微鏡偵測細胞色素C的釋出情形 60
圖 11、細胞經EtBr或ddC處理後細胞凋亡之偵測 62
圖 12、細胞經EtBr或ddC處理後再以ceramide誘發細胞凋亡之測量 63
圖 13、細胞經EtBr或ddC處理後再以CsA及ceramide處理之細胞凋亡狀況 64
圖 14、細胞凋亡定量直方圖 65
圖 15、細胞經EtBr或ddC處理後再以ceramide誘發細胞凋亡之比率 66

參考文獻 Brenner C, Grimm S. The permeability transition pore complex in cancer cell death. Oncogen 2006; 25:4744-56.
Brugg B, Michel PP, Agid Y, Ruberg M. Ceramide induces apoptosis in cultured mesencephalic neurons. J Neurochem 1996; 66:733-9.
Chauhan D, Hideshima T, Rosen S, Reed JC, Kharbanda S, Anderson KC. Apaf-1/cytochrome c-independent and Smac-dependent induction of apoptosis in multiple myeloma (MM) cells. J Biol Chem 2001; 276:24453-6.
Danial NN, Korsmeyer SJ. Cell death: critical control points. Cell 2004; 116:205-19.
Farrant M, Nusser Z. Variations on an inhibitory theme: phasic and tonic activation of GABA (A) receptors. Nat Rev Neurosci 2005; 6:215-29.
France-Lanord V, Brugg B, Michel PP, Agid Y, Ruberg M. Mitochondrial free radical signal in ceramide-dependent apoptosis: a putative mechanism for neuronal death in Parkinson's disease. J Neurochem 1997; 69:1612-21.
Goldring ES, Grossman LI, Krupnick D, Cryer DR, Marmur J. The petite mutation in yeast. Loss of mitochondrial deoxyribonucleic acid during induction of petites with ethidium bromide. J Mol Biol 1970; 52:323-35.
Grimm S, Brdiczka D. The permeability transition pore in cell death. Apoptosis 2007; 12:841-55.
Gudz TI, Tserng KY, Hoppel CL. Direct inhibition of mitochondrial respiratory chain complex III by cell-permeable ceramide. J Biol Chem 1997; 272:24154-8.
Halestrap AP, Brennerb C. The adenine nucleotide translocase: a central component of the mitochondrial permeability transition pore and key player in cell death. Curr Med Chem 2003; 10:1507-25.
Halestrap AP, Connern CP, Griffiths EJ, Kerr PM. Cyclosporin A binding to mitochondrial cyclophilin inhibits the permeability transition pore and protects hearts from ischaemia/reperfusion injury. Mol Cell Biochem 1997; 174:167-72.
Hirsch T, Marchetti P, Susin SA, Dallaporta B, Zamzami N, Marzo I, Geuskens M, Kroemer G. The apoptosis-necrosis paradox. Apoptogenic proteases activated after mitochondrial permeability transition determine the mode of cell death. Oncogene 1997; 15:1573-81.
Inoue K, Ito S, Takai D, Soejima A, Shisa H, LePecq JB, Segal-Bendirdjian E, Kagawa Y, Hayashi JI. Isolation of mitochondrial DNA-less mouse cell lines and their application for trapping mouse synaptosomal mitochondrial DNA with deletion mutations. J Biol Chem 1997; 272:15510-5.
Jacobson MD, Burne JF, King MP, Miyashita T, Reed JC, Raff MC. Bcl-2 blocks apoptosis in cells lacking mitochondrial DNA. Nature 1993; 361:365-9.
Jacotot E, Ferri KF, Kroemer G. Apoptosis and cell cycle: distinct checkpoints with overlapping upstream control. Pathol Biol 2000; 48:271-9.
Jeong SY, Seol DW. The role of mitochondria in apoptosis. BMB Rep 2008; 41:11-22.
Jia L, Bonaventura C, Bonaventura J, Stamler JS. S-nitrosohaemoglobin: a dynamic activity of blood involved in vascular control. Nature 1996; 380:221-16.
Jiang S, Cai J, Wallace DC, Jones DP. Cytochrome c-mediated apoptosis in cells lacking mitochondrial DNA. Signaling pathway involving release and caspase 3 activation is conserved. J Biol Chem 1999; 274:29905-11.
Joly V, Flandre P, Meiffredy V, Leturque N, Harel M, Aboulker JP, Yeni P. Increased risk of lipoatrophy under stavudine in HIV-1- infected patients: results of a substudy from a comparative trial. AIDS 2002; 16:2447-54.
Keswani SC, Chander B, Hasan C, Griffin JW, McArthur JC, Hoke A. FK506 is neuroprotective in a model of antiretroviral toxic neuropathy. Ann Neurol 2003; 53:57-64.
Kim JY, Kim YH, Chang I, Kim S, Pak YK, Oh BH, Yagita H, Jung YK, Oh YJ, Lee MS. Resistance of mitochondrial DNA-deficient cells to TRAIL: role of Bax in TRAIL-induced apoptosis. Oncogene 2002; 21:3139-48.
Koseki C, Herzlinger D, al-Awqati Q. Apoptosis in metanephric development. J Cell Biol 1992; 119:1327-33.
Larm JA, Vaillant F, Linnane AW, Lawen A. Up-regulation of the plasma membrane oxidoreductase as a prerequisite for the viability of human Namalwa rho0 cells. J Biol Chem 1994; 269:30097-100.
Leach TM, Karib AA, Ford EJ, Wilmshurst EC. Studies on ethidium bromide. VI. The prophylactic properties of the drug. J Comp Pathol 1995; 65:130-42.
Lee MS, Kim JY, Park SY. Resistance of rho0 cells against apoptosis. Ann N Y Acad Sci 2004; 1011:146-53.
Leibowitz RD. The effect of ethidium bromide on mitochondrial DNA synthesis and mitochondrial DNA structure in HeLa cells. J Cell Biol 1971; 51:116-22.
Lewis W, Day BJ, Copeland WC. Mitochondrial toxicity of NRTI antiviral drugs: an integrated cellular perspective. Nat Rev Drug Discov 2003; 2:812-22.
Linnane AW, Zhang C, Baumer A, Nagley P. Mitochondrial DNA mutation and the ageing process: bioenergy and pharmacological intervention. Mutat Res 1992; 275:195-208.
Lund KC, Peterson LL, Wallace KB. Absence of a universal mechanism of mitochondrial toxicity by nucleoside analogs. Antimicrob Agents Chemother 2007; 51:2531-9.
Mangoura D, Dawson G. Programmed cell death in cortical chick embryo astrocytes is associated with activation of protein kinase PK60 and ceramide formation. J Neurochem 1998; 70:130-8.
Marchetti P, Susin SA, Decaudin D, Gamen S, Castedo M, Hirsch T, Zamzami N, Naval J, Senik A, Kroemer G. Apoptosis-associated derangement of mitochondrial function in cells lacking mitochondrial DNA. Cancer Res 1996; 56:2033-8.
Mevorach D. Systemic lupus erythematosus and apoptosis: a question of balance. Clin Rev Allergy Immunol 2003; 25:49-60.
Meyer RR, Simpson MV. DNA biosynthesis in mitochondria. Differential inhibition of mitochondrial and nuclear DNA polymerases by the mutagenic dyes ethidium bromide and acriflavin. Biochem Biophys Res Commun 1969; 34:238-44.
Moraes CT, Shanske S, Tritschler HJ, Aprille JR, Andreetta F, Bonilla E, Schon EA, DiMauro S. MtDNA depletion with variable tissue expression: a novel genetic abnormality in mitochondrial. Am J Hum Genet 1991; 48:492-501.
Perlman S, Penman S. Mitochondrial protein synthesis: resistance to emetine and response to RNA synthesis inhibitors. Biochem Biophys Res Commun 1970; 40:941-8.
Peters A. A fourth type of neuroglial cell in the adult central nervous system. J Neurocytol 2004; 33345-57.
Petit C, Piétri-Rouxel F, Lesne A, Leste-Lasserre T, Mathez D, Naviaux RK, Sonigo P, Bouillaud F, Leibowitch J. Oxygen consumption by cultured human cells is impaired by a nucleoside analogue cocktail that inhibits mitochondrial DNA synthesis. Mitochondrion 2005; 5:154-61.
Reed JC, Kitada S, Kim Y, Byrd J. Modulating apoptosis pathways in low-grade B-cell malignancies using biological response modifiers. Semin Oncol 2002 ; (1 Suppl 2):10-24.
Ricci E, Moraes CT, Servidei S, Tonali P, Bonilla E, DiMauro S. Disorders associated with depletion of mitochondrial DNA. Brain Pathol 1992; 2:141-7.
Riou G, Delain E. Abnormal circular DNA molecules induced by ethidium bromide in the kinetoplast of Trypanosoma cruzi. Proc Natl Acad Sci USA 1969; 64:618-25.
Ruberg M, France-Lanord V, Brugg B, Lambeng N, Michel PP, Anglade P, Hunot S, Damier P, Faucheux B, Hirsch E, Agid Y. Neuronal death caused by apoptosis in Parkinson disease. Rev Neurol 1997; 153:499-508.
Siskind LJ, Kolesnick RN, Colombini M. Ceramide channels increase the permeability of the mitochondrial outer membrane to small proteins. J Biol Chem 2002; 277: 26796–803.
Skulachev VP Cytochrome c in the apoptotic and antioxidant cascades. FEBS Lett 1998; 423: 275–80.
Taha TA, Mullen TD, Obeid LM. A house divided: ceramide, sphingosine, and sphingosine-1-phosphate in programmed cell death. Biochim Biophys Acta 2006; 1758: 2027–36.
Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science 1995; 267:1456-62.
Veluthakal R, Palanivel R, Zhao Y, McDonald P, Gruber S, Kowluru A. Ceramide induces mitochondrial abnormalities in insulin-secreting INS-1 cells: potential mechanisms underlying ceramide-mediated metabolic dysfunction of the beta cell. Apoptosis 2005; 10:841-50.
Wang X, Zhang D. Alzheimer's disease related-genes and apoptosis. Sheng Li Ke Xue Jin Zhan 2001; 32:307-11.
White E, Sabbatini P, Debbas M, Wold WS, Kusher DI, Gooding LR. The 19-kilodalton adenovirus E1B transforming protein inhibits programmed cell death and prevents cytolysis by tumor necrosis factor alpha. Mol Cell Biol 1992; 12:2570-80.
Whiteman M, Rose P, Siau JL, Cheung NS, Tan GS, Halliwell B, Armstrong JS. Hypochlorous acid-mediated mitochondrial dysfunction and apoptosis in human hepatoma HepG2 and human fetal liver cells: role of mitochondrial permeability transition. Free Radic Biol Med 2005; 38:1571-84.
Wochna A, Niemczyk E, Kurono C, Masaoka M, Kedzior J, Slominska E, Lipinski M, Wakabayashi T. A possible role of oxidative stress in the switch mechanism of the cell death mode from apoptosis to necrosis--studies on rho0 cells. Mitochondrion 2007; 7:119-24.
Wurmb-Schwark N, Cavelier L, Cortopassi GA. A low dose of ethidium bromide leads to an increase of total mitochondrial DNA while higher concentrations induce the mtDNA 4997 deletion in a human neuronal cell line. Mutat Res 2006; 596:57-63.
Zoratti M, Szabó I. Electrophysiology of the inner mitochondrial membrane. J Bioenerg Biomembr 1994; 26:543-53.
Zoratti M, Szabò I. The mitochondrial permeability transition. Biochim Biophys Acta 1995; 1241:139-76.
Zou H, Li Y, Liu X, Wang X. An APAF-1.cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J Biol Chem 1999; 274:11549-56.
Zylber E, Penman S. Mitochondrial-associated 4 S RNA synthesis inhibition by ethidium bromide. J Mol Biol 1969; 46:201-4.

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系統識別號 U0007-2207200913112800
論文名稱(中文) 組蛋白乙醯基轉移酵素在脂多醣體刺激RAW 264.7 巨噬細胞引發環氧酵素-2表現之角色探討
論文名稱(英文) Role of Histone Acetyltransferase in Lipopolysaccharide -Induced Cyclooxygenase-2 Expression in RAW 264.7 Macrophages
校院名稱 臺北醫學大學
系所名稱(中) 醫學科學研究所
系所名稱(英) Graduate Institute of Medical Sciences
學年度 97
學期 2
出版年 98
研究生(中文) 林郡君
學號 M120096024
學位類別 碩士
語文別 中文
口試日期 2009-06-25
論文頁數 56頁
口試委員 指導教授-林建煌
共同指導教授-陳炳常
委員-蕭哲志
委員-黃聰龍
委員-嚴茂雄
關鍵字(中) 脂多醣體;組蛋白乙醯基轉移酵素;轉錄因子kB p65;環氧酵素-2;發炎
關鍵字(英) Lipopolysaccharide
Histone acetyltransferase
NF-kB p65
cyclooxygenase-2
inflammation
學科別分類
中文摘要 在過去的研究證實,內毒素是誘導巨噬細胞活化的重要因子,巨噬細胞釋放的前列腺素E (Prostaglandin E2,PGE2) 為誘導發炎反應的重要因子之一;而PGE2的生成會受到COX-2的調控。研究指出內毒素可經由HAT誘導轉錄因子活化以調控基因表現。本論文所要探討的是在RAW 264.7巨噬細胞中,組蛋白乙醯基轉移酵素 (Histone acetyltransferase, HAT) 在內毒素誘導環氧化酵素-2 (Cyclooxygenase-2,COX-2) 表現分子機轉中所扮演的角色。結果顯示在RAW 264.7巨噬細胞中,給予anacardic acid ( HAT 抑制劑) 可抑制 lipopolysaccharide (LPS) 誘導COX-2蛋白表現,使用p300 siRNA 會降低LPS 誘導COX-2蛋白表現,且發現 anacardic acid 抑制LPS會誘導 HAT 活性。我們也發現LPS會誘導 p65 及Histone H3產生乙醯化,進一步發現給予anacardic acid 可抑制 LPS 誘導 p65 乙醯化及 ?羠-luciferase 的活性。給予anacardic acid 抑制 LPS 誘導Histone H3 乙醯化。且LPS會誘導 p65 及 p300 結合在 COX-2 promoter region 。綜合以上結果發現p300將 p65 及Histone H3乙醯化在LPS誘導COX-2蛋白表現扮演重要角色,藉此研究可以提供發展控制發炎反應及敗血性休克的新方法。

英文摘要 Previous study shown that endotoxin is an important factor increased macrophage activity. Among the bioactive substances produced by macrophages including of prostaglandin E2 (PGE2) has been found the important mediators of inflammation. The PGE2 synthesis is dependent on the cyclooxygenase-2 (COX-2) expression. Previous study shown that endotoxin increased histone acetyltransferase (HAT) activity which mediates transcription factor activation and induces gene expression. In this study, we examined the role of HAT in endotoxin-induced COX-2 expression in RAW 264.7 macrophages. Pretreatment of anacardic acid (inhibitor of HAT) inhibited lipopolysaccharide (LPS)-induced COX-2 expression in a dose-dependent manner in RAW 264.7 macrophages. p300 siRNA inhibited LPS-induced COX-2 expression and anacardic acid inhibited LPS-induced HAT activity in RAW 264.7 macrophages.. We found that LPS-induced p65 acetylation and ?綑stone H3 acetylation. Furthermore, pretreatment of anacardic acid inhibited LPS-induced p65 acetylation and ?羠-luciferase activity, and Histone H3 acetylaion. Moreover, LPS increased binding of p65 and p300 to COX-2 promoter region. Taken together, we demonstrated that Histone H3 and p65 acetylation play crucial roles in LPS-induced COX-2 expression, and provided a new target for development of novel therapy in inflammation and septic shock.
論文目次 中文摘要……………………………………………………………..1
英文摘要……………………………………………………………...2
壹、 緒論……………………………………………………….3-10
貳、 實驗材料與方法
一、 實驗材料………………………………………….……..…...11
二、 實驗方法…………………………………………...……..11-15
參、 結果
一、 Anacardic acid 抑制LPS誘導RAW 264.7巨噬細胞 COX-2 蛋白表現…………………………………….……………………..16
二、 p300參與LPS所誘發RAW 264.7巨噬細胞COX-2蛋白表現………………………………………….………………………16
三、 Anacardic acid 抑制LPS所誘發RAW 264.7巨噬細胞HAT活性……………………………………………………………..16-17
四、 LPS 誘導RAW 264.7巨噬細胞 Histone H3乙醯化……………………………………………………………...……17
五、 AA 抑制LPS誘導RAW 264.7巨噬細胞 Histone H3乙醯化…………………………….………………………………….17-18
六、 LPS 誘導RAW 264.7巨噬細胞 p65蛋白乙醯化………… 18
七、 Anacardic acid 抑制LPS誘導RAW 264.7巨噬細胞 p65蛋白乙醯化………………………………………………………….. 18
八、 轉錄因子 NF-?羠 參與LPS誘導RAW 264.7巨噬細胞HAT乙醯化表現情形………...…………………………………………19
九、 LPS誘導p65與p300結合到COX-2啟動子上………………………………………………………………...…20
肆、 討論…………………...……………………………………21-25
伍、 文獻參考…………...………………………………………26-32
陸、 圖表…………………………………..…………………….33-52
參考文獻 Ait-Si-Ali, S., Polesskaya, A., Filleur, S., Ferreira, R., Duquet, A., Robin, P., Vervish, A., Trouche, D., Cabon, F., Harel-Bellan, A. (2000) CBP/p300 histone acetyl-transferase activity is important for the G1/S transition. Oncogene 19(20): 2430-2437

Akira, S., Sato, S. (2003) Toll-like receptors and their signaling mechanisms. Scand J Infect Dis 35(9): 555-562

Arany, Z., Sellers, WR., Livingston, DM., Eckner, R. (1994) E1A-associated p300 and CREB-associated CBP belong to a conserved family of coactivators. Cell 77(6): 799-800

Baeuerle, PA., Baichwal, VR. (1997) NF-kB as a frequent target for immunosuppressive and anti-inflammatory molecules. Adv Immunol 65: 111-137

Baeuerle, PA., Henkel, T. (1994) Function and activation of NF-kB in the immune system. Annu Rev Immunol 12: 141-179

Baldwin, AS., Jr. (1996) The NF-kB and IkB proteins: new discoveries and insights. Annu Rev Immunol 14: 649-683

Balk, RA., Bone, RC. (1989) The septic syndrome. Definition and clinical implications. Crit Care Clin 5(1): 1-8

Bannister, AJ., Kouzarides, T. (1996) The CBP co-activator is a histone acetyltransferase. Nature 384(6610): 641-643

Bannister, AJ., Miska, EA. (2000) Regulation of gene expression by transcription factor acetylation. Cell Mol Life Sci 57(8-9): 1184-1192

Barnes, PJ., Karin, M. (1997) Nuclear factor-kB: a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med 336(15): 1066-1071

Caivano, M., Gorgoni, B., Cohen, P., Poli, V. (2001) The induction of cyclooxygenase-2 mRNA in macrophages is biphasic and requires both CCAAT enhancer-binding protein b (C/EBP b ) and C/EBP delta transcription factors. J Biol Chem 276(52): 48693-48701

Calao, M., Burny, A., Quivy, V., Dekoninck, A., Van Lint, C. (2008) A pervasive role of histone acetyltransferases and deacetylases in an NF-kB-signaling code. Trends Biochem Sci 33(7): 339-349

Chandrasekharan, NV., Dai, H., Roos, KL., Evanson, NK., Tomsik, J., Elton, TS., Simmons, DL. (2002) COX-3, a cyclooxygenase-1 variant inhibited by acetaminophen and other analgesic/antipyretic drugs: cloning, structure, and expression. Proc Natl Acad Sci U S A 99(21): 13926-13931

Chang, YC., Li, PC., Chen, BC., Chang, MS., Wang, JL., Chiu, WT., Lin, CH. (2006) Lipoteichoic acid-induced nitric oxide synthase expression in RAW 264.7 macrophages is mediated by cyclooxygenase-2, prostaglandin E2, protein kinase A, p38 MAPK, and nuclear factor-kB pathways. Cell Signal 18(8): 1235-1243

Chen, BC., Chang, YS., Kang, JC., Hsu, MJ., Sheu, JR., Chen, TL., Teng, CM., Lin, CH. (2004) Peptidoglycan induces nuclear factor-kB activation and cyclooxygenase-2 expression via Ras, Raf-1, and ERK in RAW 264.7 macrophages. J Biol Chem 279(20): 20889-20897

Chen, CC., Wang, JK. (1999) p38 but not p44/42 mitogen-activated protein kinase is required for nitric oxide synthase induction mediated by lipopolysaccharide in RAW 264.7 macrophages. Mol Pharmacol 55(3): 481-488

Chen, LF., Mu, Y., Greene, WC. (2002) Acetylation of RelA at discrete sites regulates distinct nuclear functions of NF-kB. EMBO J 21(23): 6539-6548

Chen, LG., Hung, LY., Tsai, KW., Pan, YS., Tsai, YD., Li, YZ., Liu, YW. (2008) Wogonin, a bioactive flavonoid in herbal tea, inhibits inflammatory cyclooxygenase-2 gene expression in human lung epithelial cancer cells. Mol Nutr Food Res 52(11): 1349-1357

Chun, KS., Surh, YJ. (2004) Signal transduction pathways regulating cyclooxygenase-2 expression: potential molecular targets for chemoprevention. Biochem Pharmacol 68(6): 1089-1100

D'Acquisto, F., Iuvone, T., Rombola, L., Sautebin, L., Di Rosa, M., Carnuccio, R. (1997) Involvement of NF-kB in the regulation of cyclooxygenase-2 protein expression in LPS-stimulated J774 macrophages. FEBS Lett 418(1-2): 175-178

Dinchuk, JE., Car, BD., Focht, RJ., Johnston, JJ., Jaffee, BD., Covington, MB., Contel, NR., Eng, VM., Collins, RJ., Czerniak, PM., et al. (1995) Renal abnormalities and an altered inflammatory response in mice lacking cyclooxygenase II. Nature 378(6555): 406-409

Doetzlhofer, A., Rotheneder, H., Lagger, G., Koranda, M., Kurtev, V., Brosch, G., Wintersberger, E., Seiser, C. (1999) Histone deacetylase 1 can repress transcription by binding to Sp1. Mol Cell Biol 19(8): 5504-5511

Foxwell, B., Browne, K., Bondeson, J., Clarke, C., de Martin, R., Brennan, F., Feldmann, M. (1998) Efficient adenoviral infection with IkBa reveals that macrophage tumor necrosis factor a production in rheumatoid arthritis is NF-kB dependent. Proc Natl Acad Sci U S A 95(14): 8211-8215

Ghosh, J., Myers, CE., Jr. (1998) Arachidonic acid metabolism and cancer of the prostate. Nutrition 14(1): 48-49

Gorgoni, B., Caivano, M., Arizmendi, C., Poli, V. (2001) The transcription factor C/EBPb is essential for inducible expression of the cox-2 gene in macrophages but not in fibroblasts. J Biol Chem 276(44): 40769-40777

Grunstein, M. (1997) Histone acetylation in chromatin structure and transcription. Nature 389(6649): 349-352

Heinzel, T., Lavinsky, RM., Mullen, TM., Soderstrom, M., Laherty, CD., Torchia, J., Yang, WM., Brard, G., Ngo, SD., Davie, JR., Seto, E., Eisenman, RN., Rose, DW., Glass, CK., Rosenfeld, MG. (1997) A complex containing N-CoR, mSin3 and histone deacetylase mediates transcriptional repression. Nature 387(6628): 43-48

Hoffmann, A., Natoli, G., Ghosh, G. (2006) Transcriptional regulation via the NF-kB signaling module. Oncogene 25(51): 6706-6716

Hwang, D., Jang, BC., Yu, G., Boudreau, M. (1997) Expression of mitogen-inducible cyclooxygenase induced by lipopolysaccharide: mediation through both mitogen-activated protein kinase and NF-kB signaling pathways in macrophages. Biochem Pharmacol 54(1): 87-96

Inoue, H., Yokoyama, C., Hara, S., Tone, Y., Tanabe, T. (1995) Transcriptional regulation of human prostaglandin-endoperoxide synthase-2 gene by lipopolysaccharide and phorbol ester in vascular endothelial cells. Involvement of both nuclear factor for interleukin-6 expression site and cAMP response element. J Biol Chem 270(42): 24965-24971

Ito, K., Caramori, G., Lim, S., Oates, T., Chung, KF., Barnes, PJ., Adcock, IM. (2002) Expression and activity of histone deacetylases in human asthmatic airways. Am J Respir Crit Care Med 166(3): 392-396

Kao, HY., Ordentlich, P., Koyano-Nakagawa, N., Tang, Z., Downes, M., Kintner, CR., Evans, RM., Kadesch, T. (1998) A histone deacetylase corepressor complex regulates the Notch signal transduction pathway. Genes Dev 12(15): 2269-2277

Karin, M., Ben-Neriah, Y. (2000) Phosphorylation meets ubiquitination: the control of NF-kB activity. Annu Rev Immunol 18: 621-663

Kim, Y., Fischer, SM. (1998) Transcriptional regulation of cyclooxygenase-2 in mouse skin carcinoma cells. Regulatory role of CCAAT/enhancer-binding proteins in the differential expression of cyclooxygenase-2 in normal and neoplastic tissues. J Biol Chem 273(42): 27686-27694

Laherty, CD., Yang, WM., Sun, JM., Davie, JR., Seto, E., Eisenman, RN. (1997) Histone deacetylases associated with the mSin3 corepressor mediate mad transcriptional repression. Cell 89(3): 349-356

Lukacova, M., Barak, I., Kazar, J. (2008) Role of structural variations of polysaccharide antigens in the pathogenicity of Gram-negative bacteria. Clin Microbiol Infect 14(3): 200-206

Marks, PA., Richon, VM., Rifkind, RA. (2000) Histone deacetylase inhibitors: inducers of differentiation or apoptosis of transformed cells. J Natl Cancer Inst 92(15): 1210-1216

Mattsson, E., Verhage, L., Rollof, J., Fleer, A., Verhoef, J., van Dijk, H. (1993) Peptidoglycan and teichoic acid from Staphylococcus epidermidis stimulate human monocytes to release tumour necrosis factor-a, interleukin-1 b and interleukin-6. FEMS Immunol Med Microbiol 7(3): 281-287

Medzhitov, R., Janeway, CA., Jr. (1998) An ancient system of host defense. Curr Opin Immunol 10(1): 12-15

Merika, M., Thanos, D. (2001) Enhanceosomes. Curr Opin Genet Dev 11(2): 205-208

Mitchell, JA., Larkin, S., Williams, TJ. (1995) Cyclooxygenase-2: regulation and relevance in inflammation. Biochem Pharmacol 50(10): 1535-1542

Miyamoto, S., Verma, IM. (1995) Rel/NF-kB/IkB story. Adv Cancer Res 66: 255-292

Ng, HH., Bird, A. (1999) DNA methylation and chromatin modification. Curr Opin Genet Dev 9(2): 158-163

Ogryzko, VV., Schiltz, RL., Russanova, V., Howard, BH., Nakatani, Y. (1996) The transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell 87(5): 953-959

Park, GY., Joo, M., Pedchenko, T., Blackwell, TS., Christman, JW. (2004) Regulation of macrophage cyclooxygenase-2 gene expression by modifications of histone H3. Am J Physiol Lung Cell Mol Physiol 286(5): L956-962

Pedersen, MO., Pedersen, DS., Pedersen, M., Penkowa, M. (2008) [Neisseria meningitidis. The pathophysiological role of lipopolysaccharides in association with meningococcal disease and septic shock]. Ugeskr Laeger 170(6): 421-426

Puri, PL., Sartorelli, V., Yang, XJ., Hamamori, Y., Ogryzko, VV., Howard, BH., Kedes, L., Wang, JY., Graessmann, A., Nakatani, Y., Levrero, M. (1997) Differential roles of p300 and PCAF acetyltransferases in muscle differentiation. Mol Cell 1(1): 35-45

Rogers, HJ. (1974) Peptidoglycans (mucopeptides): structure, function, and variations. Ann N Y Acad Sci 235(0): 29-51

Roth, SY., Denu, JM., Allis, CD. (2001) Histone acetyltransferases. Annu Rev Biochem 70: 81-120

Schiltz, RL., Mizzen, CA., Vassilev, A., Cook, RG., Allis, CD., Nakatani Y (1999) Overlapping but distinct patterns of histone acetylation by the human coactivators p300 and PCAF within nucleosomal substrates. J Biol Chem 274(3): 1189-1192

Schmeck, B., Beermann, W., van Laak, V., Zahlten, J., Opitz, B., Witzenrath, M., Hocke, AC., Chakraborty, T., Kracht, M., Rosseau, S., Suttorp, N., Hippenstiel, S. (2005) Intracellular bacteria differentially regulated endothelial cytokine release by MAPK-dependent histone modification. J Immunol 175(5): 2843-2850

Sen, R., Baltimore, D. (1986) Inducibility of k immunoglobulin enhancer-binding protein Nf-kB by a posttranslational mechanism. Cell 47(6): 921-928

Siebenlist, U., Franzoso, G., Brown, K. (1994) Structure, regulation and function of NF-kB. Annu Rev Cell Biol 10: 405-455

Solomkin, JS. (2001) Antibiotic resistance in postoperative infections. Crit Care Med 29(4 Suppl): N97-99

Sterner, DE., Berger, SL. (2000) Acetylation of histones and transcription-related factors. Microbiol Mol Biol Rev 64(2): 435-459

Strahl, BD., Allis, CD. (2000) The language of covalent histone modifications. Nature 403(6765): 41-45

Thompson, PR., Kurooka, H., Nakatani, Y., Cole, PA. (2001) Transcriptional coactivator protein p300. Kinetic characterization of its histone acetyltransferase activity. J Biol Chem 276(36): 33721-33729

Wade, PA., Pruss, D., Wolffe, AP. (1997) Histone acetylation: chromatin in action. Trends Biochem Sci 22(4): 128-132

Williams, JA., Shacter, E. (1997) Regulation of macrophage cytokine production by prostaglandin E2. Distinct roles of cyclooxygenase-1 and -2. J Biol Chem 272(41): 25693-25699

Yang, KH., Lee, MG. (2008) Effects of endotoxin derived from Escherichia coli lipopolysaccharide on the pharmacokinetics of drugs. Arch Pharm Res 31(9): 1073-1086












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系統識別號 U0007-2507200819302000
論文名稱(中文) 人參及人參皂苷Rg1與N-乙醯半胱胺酸在順氯氨鉑引發的腎毒性於純系小鼠的藥效評估
論文名稱(英文) Effects of ginseng , ginsenoside Rg1 and N-acetylcysteine on cisplatin-induced nephrotoxicity in inbred mice
校院名稱 臺北醫學大學
系所名稱(中) 藥學研究所
系所名稱(英) Graduate Institute of Pharmacy
學年度 96
學期 2
出版年 97
研究生(中文) 黃致云
學號 M301095018
學位類別 碩士
語文別 中文
口試日期 2008-06-20
論文頁數 98頁
口試委員 指導教授-陳世銘
委員-方嘉佑
委員-許秀蘊
關鍵字(中) Cisplatin腎毒性
人參
人參皂苷
N-乙醯半胱胺酸
甲型腫瘍壞死因子
p21
增殖細胞核抗原
關鍵字(英) Cisplatin nephrotoxicity
ginseng
ginsenosides
N-acetylcysteine
TNF-alpha
p21
PCNA
學科別分類
中文摘要 順氯氨鉑(cisplatin, CDDP)是臨床上治療固體癌的常用化學治療藥物,其所引起的腎毒性常是限制臨床使用的主要原因。本研究的目的即在於評估人參及其純成分人參皂苷與N-乙醯半胱胺酸(N-acetylcysteine)作為預防藥物於CDDP所引起的腎炎之預防效果。實驗以6週齡雌鼠(BALB/c mice, female),經腹腔連續五天給予CDDP 5 mg/kg/d以引發CDDP腎炎。在給予CDDP前五天開始經口單獨投予小鼠人參濃縮劑(ginseng extract,GE) 250 mg/kg/d或人參皂苷(ginsengoside,GS) Rg1 5 mg/kg/d及並分別合併N-acetylcysteine 450 mg/kg/d作為預防藥物。實驗結果顯示,給予GE、GS Rg1及N-acetylcysteine對於N-acetyl-beta-D-glucosaminidase (NAG) 、尿中肌酸酐(urine creatinine)、尿蛋白(urine protein)與血中尿素氮(BUN)皆有不同程度的改善效果;腎組織損傷相較於對照組也有減緩的趨勢。在免疫螢光染色方面,TNF-alpha(tumor necrosis factor-alpha)的量明顯受到抑制,p21及PCNA(proliferating cell nuclear antigen)的表現亦有不同程度的增加。綜合實驗結果,合併治療組對於預防CDDP所引起的腎毒性效果最佳。因此可以推論,經口投予人參濃縮劑、人參皂苷Rg1、N-acetylcysteine可以藉由抑制發炎反應、阻止細胞週期的前進並促進DNA修復以達到腎臟保護的效果。
英文摘要 Cisplatin (CDDP) is one of the most commonly used antineoplastic agents for the solid tumor treatment. The major side effect of CDDP is nephrotoxicity. It is dose-related and has become a chief limitation of its clinical use. The purpose of this study was to evaluate the preventive effects of ginseng extract (GE), its active component, ginsenoside Rg1(GS Rg1) and N-acetylcysteine (NAC) on CDDP-induced nephrotoxicity in bred mice. Six-week-old female BALB/c mice were administered with 5 mg/kg of CDDP intraperitoneally once daily for 5 days. 250 mg/kg of GE or 5 mg/kg of GS Rg1 combination with 450 mg/kg/d of NAC were given orally once a day from 5 days before CDDP administration. Urinary N-acetyl-??-D-glucosaminidase (NAG), urinary creatinine(Ucr) and blood urea nitrogen (BUN) were determined, Renal tissues were served to histological examination. The antibodies including tumor necrosis factor-alpha(TNF-alpha), p21 and proliferating cell nuclear antigen (PCNA) was chosen to recognize the specific antigens that deposited in injury sites. Our findings demonstrated that GE, GS Rg1 and NAC attenuate CDDP-induced nephrotixicity by inhibiting TNF-alpha expression and inducing cell cycle arrest to repair DNA damage.According to this study, the effect of combination treatment was superior to other group.
論文目次 目錄 I
圖目錄 V
表目錄 VII
縮寫表 i
中文摘要 ii
空Abstract iv
第一章 緒言 1
第二章 文獻回顧 4
第一節 順氯氨鉑(Cisplatin)的作用機轉及臨床使用 4
第二節 Cisplatin引起腎毒性的臨床表徵與機轉 10
第三節 Cisplatin與細胞週期的調控 17
第四節 人參及其藥效研究 25
4.1 生藥學的考察 25
4.2 人參的藥效研究 27
第五節 人參皂苷Rg1之藥效研究 31
5.1 人參皂苷之結構特性 31
5.2人參皂苷Rg1之藥效研究 34
第六節 N-乙醯半胱胺酸 N-acetylcysteine 36
第三章 研究目的 39
第四章 材料與方法 40
第一節 人參濃縮劑與N-乙醯半胱胺酸在CDDP引起腎炎模型的藥效評估 40
1.1實驗動物 40
1.2實驗藥物 40
1.3人參濃縮劑與N-乙醯半胱胺酸在此腎炎模型之實驗設計 41
1.4尿液收集 41
1.5動物犧牲法、血液及組織切片製作 41
1.6尿中NAG、Creatinine及蛋白的含量分析 42
1.7血清中BUN值的含量測定 43
1.8 Periodoic acid-Schiff’s(PAS)stain組織染色 43
1.9組織損傷程度的量化 44
1.10免疫螢光染色(Immunofluorescence) 44
1.11統計方法 45
第二節 人參皂苷Rg1與N-乙醯半胱胺酸在CDDP引起腎炎模型的藥效評估 47
2.1實驗動物 47
2.2實驗藥物 47
2.3人參皂苷合併N-乙醯半胱胺酸在此腎炎模型之實驗設計 47
2.4尿液收集 48
2.5動物犧牲法、血液及組織切片製作 48
2.6尿中NAG、Creatinine及蛋白的含量分析 48
2.7血清中BUN值的含量測定 48
2.8 Periodoic acid-Schiff’s(PAS) stain組織染色 48
2.9組織損傷程度的量化 48
2.10免疫螢光染色 48
2.11統計方法 48
第五章 結果 50
第一節 人參濃縮劑與N-乙醯半胱胺酸在CDDP引起腎炎模型的藥效評估 50
1.1 尿中NAG、Creatinine及蛋白的含量分析 50
1.2 血清中BUN分析 51
1.3 組織病理PAS染色 51
1.4組織損傷量化分析 52
1.5免疫螢光染色 52
第二節 人參皂苷Rg1及N-乙醯半胱胺酸在CDDP引起腎炎模型的藥效評估 62
2.1 尿中NAG、Creatinine及蛋白的含量分析 62
2.2 血清中BUN分析 63
2.3 組織病理PAS染色 63
2.4組織損傷量化分析 64
2.5免疫螢光染色 64
第六章 討論 74
第一節 人參濃縮劑及人參皂苷Rg1在CDDP引起腎炎模型的藥效評估空. 74
第二節 N-乙醯半胱胺酸在CDDP引起腎炎模型的藥效評估 78
第三節 人參濃縮劑及人參皂苷Rg1合併N-乙醯半胱胺酸在CDDP引起腎炎模型的藥效評估 81
第七章 結論 84
參考文獻 86

參考文獻 中華民國衛生署. 中華民國九十五年臺灣地區死因統計結果摘要 http://www.doh.gov.tw/statistic/index.htm. Accessed June 11th, 2007.
2. 世界衛生組織. Cancer: diet and physical activity's impact. Accessed May 28th, 2008.
3. CISPLATIN.MICROMEDEX(R) Healthcare Series: Thomson Healthcare.; 2008. Accessed.
4. Rosenberg B, Vancamp L, Krigas T. Inhibition of cell division in excherichia coli by electrolysis products from a platinum ecectrode Nature.1965;205:698-699.
5. Jordan P, Carmo-Fonseca M. Molecular mechanisms involved in cisplatin cytotoxicity. Cellular & Molecular Life Sciences. 2000;57(8-9):1229-1235.
6. Muggia FM. Cisplatin update. Seminars in Oncology.1991;18(1 Suppl 3):1-4.
7. Leng M, Brabec V. DNA adducts of cisplatin, transplatin and platinum-intercalating drugs. IARC Scientific Publications. 1994(125):339-348.
8. Ries F, Klastersky J. Nephrotoxicity induced by cancer chemotherapy with special emphasis on cisplatin toxicity. American Journal of Kidney Diseases.1986;8(5):368-379.
9. Leibbrandt ME, Wolfgang GH, Metz AL, Ozobia AA, Haskins JR. Critical subcellular targets of cisplatin and related platinum analogs in rat renal proximal tubule cells. Kidney International. 1995;48(3):761-770.
10. Chu G. Cellular responses to cisplatin. The roles of DNA-binding proteins and DNA repair. Journal of Biological Chemistry. 1994;269(2):787-790.
11. Schrier RW. Diseases of the kidney and urinary tract. Vol 2. 7th ed ed: Philadelphia, PA, USA : Lippincott Williams & Wilkins; 2001.
12. Raymond J.M N, Jonh de vries, Mannfred A. Hollinger. Toxicologie: CRC press 1996.
13. Abeloff MD. Clinical Oncology 3rd ed. New York Churchill Livingstone; 2004.
14. de Jongh FE, van Veen RN, Veltman SJ, de Wit R, van der Burg ME, van den Bent MJ, Planting AS, Graveland WJ, Stoter G, Verweij J. Weekly high-dose cisplatin is a feasible treatment option: analysis on prognostic factors for toxicity in 400 patients. British Journal of Cancer. 2003;88(8):1199-1206.
15. Skeel RT. Handbook of Cancer Chemotherapy. Fifth ed; 2007.
16. Hartmann JT, Knop S, Fels LM, van Vangerow A, Stolte H, Kanz L, Bokemeyer C. The use of reduced doses of amifostine to ameliorate nephrotoxicity of cisplatin/ifosfamide-based chemotherapy in patients with solid tumors. Anti-Cancer Drugs. 2000;11(1):1-6.
17. Santini V, Giles FJ. The potential of amifostine: from cytoprotectant to therapeutic agent. Haematologica. 1999;84(11):1035-1042.
18. Hartmann JT, Fels LM, Knop S, Stolt H, Kanz L, Bokemeyer C. A randomized trial comparing the nephrotoxicity of cisplatin/ifosfamide-based combination chemotherapy with or without amifostine in patients with solid tumors. Invest New Drugs. 2000;18(3):281-289.
19. Kuhlmann MK, Burkhardt G, Kohler H. Insights into potential cellular mechanisms of cisplatin nephrotoxicity and their clinical application. Nephrology Dialysis Transplantation. 1997;12(12):2478-2480.
20. Schuchter LM, Hensley ML, Meropol NJ, Winer EP, American Society of Clinical Oncology Chemotherapy and Radiotherapy Expert P. 2002 update of recommendations for the use of chemotherapy and radiotherapy protectants: clinical practice guidelines of the American Society of Clinical Oncology. Journal of Clinical Oncology.2002;20(12):2895-2903.
21. Daugaard G, Abildgaard U. Cisplatin nephrotoxicity. A review. Cancer Chemotherapy & Pharmacology. 1989;25(1):1-9.
22. Choie DD, Longnecker DS, del Campo AA. Acute and chronic cisplatin nephropathy in rats. Laboratory Investigation. 1981;44(5):397-402.
23. Safirstein R, Winston J, Moel D, Dikman S, Guttenplan J. Cisplatin nephrotoxicity: insights into mechanism. International Journal of Andrology. 1987;10(1):325-346.
24. Meyer KB, Madias NE. Cisplatin nephrotoxicity. Mineral & Electrolyte Metabolism. 1994;20(4):201-213.
25. Litterst CL TI, Guarino AM. Plasma levels and organ distribution of platinum in the rat, dog, and dog fish following intravenous administration of cis-DDP(ll) J Clin Hemat Oncol. 1977;7:169.
26. Alex M. Davison JSC, Jean-pierre Grunfeld, David N.S. Kerr, Eberhard Ritz, Christopher G.Winearls. Oxford Textbook of Clinical Nephrology. Vol 3. 2th edition ed: Oxford ; New York : Oxford University Press; 1998.
27. Berns JS, Ford PA. Renal toxicities of antineoplastic drugs and bone marrow transplantation. Seminars in Nephrology. 1997;17(1):54-66.
28. Schilsky RL, Anderson T. Hypomagnesemia and renal magnesium wasting in patients receiving cisplatin. Annals of Internal Medicine. 1979;90(6):929-931.
29. Lam M, Adelstein DJ. Hypomagnesemia and renal magnesium wasting in patients treated with cisplatin. American Journal of Kidney Diseases. 1986;8(3):164-169.
30. Sutton RA, Walker VR, Halabe A, Swenerton K, Coppin CM. Chronic hypomagnesemia caused by cisplatin: effect of calcitriol. Journal of Laboratory & Clinical Medicine. 1991;117(1):40-43.
31. Winston JA, Safirstein R. Reduced renal blood flow in early cisplatin-induced acute renal failure in the rat. American Journal of Physiology. 1985;249(4 Pt 2):F490-496.
32. Safirstein R, Miller P, Dikman S, Lyman N, Shapiro C. Cisplatin nephrotoxicity in rats: defect in papillary hypertonicity. American Journal of Physiology. 1981;241(2):F175-185.
33. Arany I, Safirstein RL. Cisplatin nephrotoxicity. Seminars in Nephrology. 2003;23(5):460-464.
34. Lau AH. Apoptosis induced by cisplatin nephrotoxic injury. Kidney International. 1999;56(4):1295-1298.
35. Dobyan DC, Levi J, Jacobs C, Kosek J, Weiner MW. Mechanism of cis-platinum nephrotoxicity: II. Morphologic observations. Journal of Pharmacology & Experimental Therapeutics. 1980;213(3):551-556.
36. Ross DA, Gale GR. Reduction of the renal toxicity of cis-dichlorodiammineplatinum(II) by probenecid. Cancer Treatment Reports. 1979;63(5):781-787.
37. Weiner MW, Jacobs C. Mechanism of cisplatin nephrotoxicity. Federation Proceedings. 1983;42(13):2974-2978.
38. Fatima S, Yusufi AN, Mahmood R. Effect of cisplatin on renal brush border membrane enzymes and phosphate transport. Human & Experimental Toxicology. 2004;23(12):547-554.
39. Tsuruya K, Tokumoto M, Ninomiya T, Hirakawa M, Masutani K, Taniguchi M, Fukuda K, Kanai H, Hirakata H, Iida M. Antioxidant ameliorates cisplatin-induced renal tubular cell death through inhibition of death receptor-mediated pathways. American Journal of Physiology - Renal Physiology. 2003;285(2):F208-218.
40. Levi J, Jacobs C, Kalman SM, McTigue M, Weiner MW. Mechanism of cis-platinum nephrotoxicity: I. Effects of sulfhydryl groups in rat kidneys. Journal of Pharmacology & Experimental Therapeutics. 1980;213(3):545-550.
41. Ramesh G, Reeves WB. Inflammatory cytokines in acute renal failure. Kidney International - Supplement. 2004(91):S56-61.
42. Kaushal GP, Kaushal V, Hong X, Shah SV. Role and regulation of activation of caspases in cisplatin-induced injury to renal tubular epithelial cells. Kidney International. 2001;60(5):1726-1736.
43. Nowak G. Protein kinase C-alpha and ERK1/2 mediate mitochondrial dysfunction, decreases in active Na+ transport, and cisplatin-induced apoptosis in renal cells. Journal of Biological Chemistry. 2002;277(45):43377-43388.
44. Rosenberg JM, Sato PH. Cisplatin inhibits in vitro translation by preventing the formation of complete initiation complex. Molecular Pharmacology. 1993;43(3):491-497.
45. Courjault-Gautier F, Le Grimellec C, Giocondi MC, Toutain HJ. Modulation of sodium-coupled uptake and membrane fluidity by cisplatin in renal proximal tubular cells in primary culture and brush-border membrane vesicles. Kidney International. 1995;47(4):1048-1056.
46. Bompart G. Cisplatin-induced changes in cytochrome P-450, lipid peroxidation and drug-metabolizing enzyme activities in rat kidney cortex. Toxicology Letters. 1989;48(2):193-199.
47. Mistry P, Merazga Y, Spargo DJ, Riley PA, McBrien DC. The effects of cisplatin on the concentration of protein thiols and glutathione in the rat kidney. Cancer Chemotherapy & Pharmacology. 1991;28(4):277-282.
48. Galle J. Oxidative stress in chronic renal failure. Nephrology Dialysis Transplantation. 2001;16(11):2135-2137.
49. Shackelford RE, Kaufmann WK, Paules RS. Oxidative stress and cell cycle checkpoint function. Free Radical Biology & Medicine. May 1 2000;28(9):1387-1404.
50. Ichikawa I, Kiyama S, Yoshioka T. Renal antioxidant enzymes: their regulation and function. Kidney International. 1994;45(1):1-9.
51. Halliwell B. The role of oxygen radicals in human disease, with particular reference to the vascular system. Haemostasis. 1993;23 Suppl 1:118-126.
52. Halliwell B. Antioxidant defence mechanisms: from the beginning to the end (of the beginning). Free Radical Research. 1999;31(4):261-272.
53. Klahr S. Oxygen radicals and renal diseases. Mineral & Electrolyte Metabolism. 1997;23(3-6):140-143.
54. Sugiyama S, Hayakawa M, Kato T, Hanaki Y, Shimizu K, Ozawa T. Adverse effects of anti-tumor drug, cisplatin, on rat kidney mitochondria: disturbances in glutathione peroxidase activity. Biochemical & Biophysical Research Communications. 1989;159(3):1121-1127.
55. Schrier RW. Cancer therapy and renal injury. Journal of Clinical Investigation. 2002;110(6):743-745.
56. Goossens V, Grooten J, De Vos K, Fiers W. Direct evidence for tumor necrosis factor-induced mitochondrial reactive oxygen intermediates and their involvement in cytotoxicity. Proceedings of the National Academy of Sciences of the United States of America. 1995;92(18):8115-8119.
57. Ramesh G, Reeves WB. TNFR2-mediated apoptosis and necrosis in cisplatin-induced acute renal failure. American Journal of Physiology - Renal Physiology. 2003;285(4):F610-618.
58. Ramesh G, Reeves WB, Ramesh G, Reeves WB. TNF-alpha mediates chemokine and cytokine expression and renal injury in cisplatin nephrotoxicity. Journal of Clinical Investigation. 2002;110(6):835-842.
59. 丁明孝等編著. 細胞分子生物學. 第一版. 台北市: 九州圖書文物有限公司; 2001.
60. Lewin B. Genes VIII: Pearson Prentice Hall; 2004.
61. Price PM, Megyesi J, Safirstein RL. Cell cycle regulation: repair and regeneration in acute renal failure. Seminars in Nephrology. 2003;23(5):449-459.
62. Megyesi J, Udvarhelyi N, Safirstein RL, Price PM. The p53-independent activation of transcription of p21 WAF1/CIP1/SDI1 after acute renal failure. American Journal of Physiology. 1996;271(6):F1211-1216.
63. Megyesi J, Andrade L, Vieira JM, Jr., Safirstein RL, Price PM. Positive effect of the induction of p21WAF1/CIP1 on the course of ischemic acute renal failure. Kidney International. 2001;60(6):2164-2172.
64. Nowak G, Price PM, Schnellmann RG. Lack of a functional p21WAF1/CIP1 gene accelerates caspase-independent apoptosis induced by cisplatin in renal cells. American Journal of Physiology - Renal Physiology. 2003;285(3):F440-450.
65. Megyesi J, Safirstein RL, Price PM, Megyesi J, Safirstein RL, Price PM. Induction of p21WAF1/CIP1/SDI1 in kidney tubule cells affects the course of cisplatin-induced acute renal failure. Journal of Clinical Investigation. 1998;101(4):777-782.
66. Yasuda H, Kato A, Miyaji T, Zhou H, Togawa A, Hishida A. Insulin-like growth factor-I increases p21 expression and attenuates cisplatin-induced acute renal injury in rats. Clinical & Experimental Nephrology. 2004;8(1):27-35.
67. Price PM, Safirstein RL, Megyesi J. Protection of renal cells from cisplatin toxicity by cell cycle inhibitors. American Journal of Physiology - Renal Physiology. 2004;286(2):F378-384.
68. Zhou H, Kato A, Yasuda H, Miyaji T, Fujigaki Y, Yamamoto T, Yonemura K, Hishida A. The induction of cell cycle regulatory and DNA repair proteins in cisplatin-induced acute renal failure. Toxicology & Applied Pharmacology. 2004;200(2):111-120.
69. Miyaji T, Kato A, Yasuda H, Fujigaki Y, Hishida A. Role of the increase in p21 in cisplatin-induced acute renal failure in rats. Journal of the American Society of Nephrology. 2001;12(5):900-908.
70. Lin Z, Lim S, Viani MA, Sapp M, Lim MS. Down-regulation of telomerase activity in malignant lymphomas by radiation and chemotherapeutic agents. American Journal of Pathology. 2001;159(2):711-719.
71. Shankland SJ, Wolf G, Shankland SJ, Wolf G. Cell cycle regulatory proteins in renal disease: role in hypertrophy, proliferation, and apoptosis. American Journal of Physiology - Renal Physiology. 2000;278(4):F515-529.
72. Benjamin L. Genes VII. 2000.
73. Kelman Z. PCNA: structure, functions and interactions. Oncogene. 1997;14(6):629-640.
74. Miyachi K, Fritzler MJ, Tan EM. Autoantibody to a nuclear antigen in proliferating cells. Journal of Immunology. 1978;121(6):2228-2234.
75. Bravo R, Celis JE. A search for differential polypeptide synthesis throughout the cell cycle of HeLa cells. Journal of Cell Biology. 1980;84(3):795-802.
76. Maga G, Hubscher U. Proliferating cell nuclear antigen (PCNA): a dancer with many partners. Journal of Cell Science. 2003;116(15):3051-3060.
77. Nakajima T, Miyaji T, Kato A, Ikegaya N, Yamamoto T, Hishida A. Uninephrectomy reduces apoptotic cell death and enhances renal tubular cell regeneration in ischemic ARF in rats. American Journal of Physiology. 1996;271(4):F846-853.
78. Sano K, Fujigaki Y, Miyaji T, Ikegaya N, Ohishi K, Yonemura K,Hishida A. Role of apoptosis in uranyl acetate-induced acute renal failure and acquired resistance to uranyl acetate. Kidney International. 2000;57(4):1560-1570.
79. McCormick D, Hall PA, McCormick D, Hall PA. The complexities of proliferating cell nuclear antigen. Histopathology. 1992;21(6):591-594.
80. Celis JE, Madsen P, Celis JE, Madsen P. Increased nuclear cyclin/PCNA antigen staining of non S-phase transformed human amnion cells engaged in nucleotide excision DNA repair. FEBS Letters. 1986;209(2):277-283.
81. 賴榮祥. 原色生藥學. 台中市: 創譯出版社; 2000.
82. Attele AS, Wu JA, Yuan CS. Ginseng pharmacology: multiple constituents and multiple actions. Biochemical Pharmacology. 1999;58(11):1685-1693.
83. Liu H, Baliga R. Cytochrome P450 2E1 null mice provide novel protection against cisplatin-induced nephrotoxicity and apoptosis. Kidney International. 2003;63(5):1687-1696.
84. Kiefer D, Pantuso T. Panax ginseng. American Family Physician. 2003;68(8):1539-1542.
85. Kitts D, Hu C. Efficacy and safety of ginseng. Public Health Nutrition. 2000;3(4A):473-485.
86. Chang YS, Seo EK, Gyllenhaal C, Block KI. Panax ginseng: a role in cancer therapy? Integrative Cancer Therapies. 2003;2(1):13-33.
87. Shin HR, Kim JY, Yun TK, Morgan G, Vainio H. The cancer-preventive potential of Panax ginseng: a review of human and experimental evidence. Cancer Causes & Control. 2000;11(6):565-576.
88. Ong YC, Yong EL. Panax (ginseng)--panacea or placebo? Molecular and cellular basis of its pharmacological activity. Annals of the Academy of Medicine, Singapore. 2000;29(1):42-46.
89. Gillis CN. Panax ginseng pharmacology: a nitric oxide link? Biochemical Pharmacology. 1997;54(1):1-8.
90. Vogler BK, Pittler MH, Ernst E. The efficacy of ginseng. A systematic review of randomised clinical trials. European Journal of Clinical Pharmacology. 1999;55(8):567-575.
91. Rimar S, Lee-Mengel M, Gillis CN. Pulmonary protective and vasodilator effects of a standardized Panax ginseng preparation following artificial gastric digestion. Pulmonary Pharmacology. 1996;9(4):205-209.
92. Voces J, Alvarez AI, Vila L, Ferrando A, Cabral de Oliveira C, Prieto JG. Effects of administration of the standardized Panax ginseng extract G115 on hepatic antioxidant function after exhaustive exercise. Comparative Biochemistry & Physiology Part C Pharmacology, Toxicology, Endocrinology. 1999;123(2):175-184.
93. Sotaniemi EA, Haapakoski E, Rautio A. Ginseng therapy in non-insulin-dependent diabetic patients. Diabetes Care. 1995;18(10):1373-1375.
94. Jeong TC, Kim HJ, Park JI, Ha CS, Park JD, Kim SI, Roh JK. Protective effects of red ginseng saponins against carbon tetrachloride-induced hepatotoxicity in Sprague Dawley rats. Planta Medica. 1997;63(2):136-140.
95. Cho JY, Yoo ES, Baik KU, Park MH, Han BH. In vitro inhibitory effect of protopanaxadiol ginsenosides on tumor necrosis factor (TNF)-alpha production and its modulation by known TNF-alpha antagonists. Planta Medica. 2001;67(3):213-218.
96. Cheng Y, Shen LH, Zhang JT. Anti-amnestic and anti-aging effects of ginsenoside Rg1 and Rb1 and its mechanism of action. Acta Pharmacologica Sinica. Feb 2005;26(2):143-149.
97. Chen XC, Zhou YC, Chen Y, Zhu YG, Fang F, Chen LM. Ginsenoside Rg1 reduces MPTP-induced substantia nigra neuron loss by suppressing oxidative stress. Acta Pharmacologica Sinica. 2005;26(1):56-62.
98. Chen XC, Zhu YG, Zhu LA, Huang C, Chen Y, Chen LM, Fang F, Zhou YC, Zhao CH. Ginsenoside Rg1 attenuates dopamine-induced apoptosis in PC12 cells by suppressing oxidative stress. European Journal of Pharmacology. 2003;473(1):1-7.
99. Chen XC, Fang F, Zhu YG, Chen LM, Zhou YC, Chen Y. Protective effect of ginsenoside Rg1 on MPP+-induced apoptosis in SHSY5Y cells. Journal of Neural Transmission. 2003;110(8):835-845.
100. Leung KW, Cheng YK, Mak NK, Chan KK, Fan TP, Wong RN. Signaling pathway of ginsenoside-Rg1 leading to nitric oxide production in endothelial cells. FEBS Lett. 2006;580(13):3211-3216.
101. Lu JP, Ma ZC, Yang J, Huang J, Wang SR, Wang SQ. Ginsenoside Rg1-induced alterations in gene expression in TNF-alpha stimulated endothelial cells. Chinese Medical Journal. 2004;117(6):871-876.
102. Zhang HS, Wang SQ. Ginsenoside Rg1 inhibits tumor necrosis factor-alpha (TNF-alpha)-induced human arterial smooth muscle cells (HASMCs) proliferation. Journal of Cellular Biochemistry. 2006;98(6):1471-1481.
103. Wu CF, Bi XL, Yang JY, Zhan JY, Dong YX, Wang JH, Wang JM, Zhang R, Li X. Differential effects of ginsenosides on NO and TNF-alpha production by LPS-activated N9 microglia. Int Immunopharmacol. 2007;7(3):313-320.
104. Alfred Goodman Gliman TWR, Palmer Taylor. Goodman&Gliman's The Pharmacological Basis of Therapeutics(ed, Insel,PA): Macmillan Publishing Company; 1990.
105. Holdiness MR. Clinical pharmacokinetics of N-acetylcysteine. Clin Pharmacokinet. 1991;20(2):123-134.
106. Product Information: Acetylcysteine Solution, acetylcysteine solution, USP. Columbus, OH, USA: Roxane Laboratories, Inc; 2001.
107. Peterson RG, Rumack BH. Treating acute acetaminophen poisoning with acetylcysteine. JAMA. 1977;237(22):2406-2407.
108. Safirstein R, Andrade L, Vieira JM. Acetylcysteine and nephrotoxic effects of radiographic contrast agents--a new use for an old drug. N Engl J Med. Jul 20 2000;343(3):210-212.
109. Rumack BH, Peterson RG. Acetaminophen toxicity. West J Med. 1980;132(1):61.
110. Peterson RG, Rumack BH. Toxicity of acetaminophen overdose. JACEP. 1978;7(5):202-205.
111. Peterson RG, Rumack BH. N-acetylcysteine for acetaminophen overdosage (cont.). N Engl J Med. 1977;296(9):515.
112. Tepel M, van der Giet M, Schwarzfeld C, Laufer U, Liermann D, Zidek W. Prevention of radiographic-contrast-agent-induced reductions in renal function by acetylcysteine. N Engl J Med. 2000;343(3):180-184.
113. Salahudeen A, Poovala V, Parry W, Pande R,Kanji V, Ansari N, Morrow J, Roberts J. Cisplatin induces N-acetyl cysteine suppressible F2-isoprostane production and injury in renal tubular epithelial cells. Journal of the American Society of Nephrology. 1998;9(8):1448-1455.
114. DiMari J, Megyesi J, Udvarhelyi N, Price P, Davis R, Safirstein R. N-acetyl cysteine ameliorates ischemic renal failure. Am J Physiol. 1997;272(3 Pt 2):F292-298.
115. Salom MG, Ramirez P, Carbonell LF, Lopez Conesa E, Cartagena J, Quesada T, Parrilla P, Fenoy FJ. Protective effect of N-acetyl-L-cysteine on the renal failure induced by inferior vena cava occlusion. Transplantation. 1998;65(10):1315-1321.
116. Yano T, Itoh Y, Matsuo M, Kawashiri T, Egashira N, Oishi R. Involvement of both tumor necrosis factor-alpha-induced necrosis and p53-mediated caspase-dependent apoptosis in nephrotoxicity of cisplatin. Apoptosis. 2007;12(10):1901-1909.
117. Sheikh-Hamad D, Timmins K, Jalali Z. Cisplatin-induced renal toxicity: possible reversal by N-acetylcysteine treatment. Journal of the American Society of Nephrology. 1997;8(10):1640-1644.
118. Appenroth D, Winnefeld K, Schroter H, Rost M. Beneficial effect of acetylcysteine on cisplatin nephrotoxicity in rats. J Appl Toxicol. 1993;13(3):189-192.
119. Appenroth D, Braunlich H. Age differences in cisplatinum nephrotoxicity. Toxicology. 1984;32(4):343-353.
120. Appenroth D, Winnefeld K. Role of glutathione for cisplatin nephrotoxicity in young and adult rats. Ren Fail. 1993;15(2):135-139.
121. Mishima K, Baba A, Matsuo M, Itoh Y, Oishi R. Protective effect of cyclic AMP against cisplatin-induced nephrotoxicity. Free Radical Biology & Medicine. 2006;40(9):1564-1577.
122. Babu E, Gopalakrishnan VK, Sriganth IN, Gopalakrishnan R, Sakthisekaran D. Cisplatin induced nephrotoxicity and the modulating effect of glutathione ester. Mol Cell Biochem. 1995;144(1):7-11.
123. Ramesh G, Reeves WB. Salicylate reduces cisplatin nephrotoxicity by inhibition of tumor necrosis factor-alpha. Kidney Int. 2004;65(2):490-499.
124. Ramesh G, Reeves WB. Inflammatory cytokines in acute renal failure. Kidney Int Suppl. 2004(91):S56-61.
125. Ramesh G, Reeves WB. p38 MAP kinase inhibition ameliorates cisplatin nephrotoxicity in mice. Am J Physiol Renal Physiol. 2005;289(1):F166-174.
126. Kim YW, Song DK, Kim WH, Lee KM, Wie MB, Kim YH, Kee SH, Cho MK. Long-term oral administration of ginseng extract decreases serum gamma-globulin and IgG1 isotype in mice. Journal of Ethnopharmacology. 1997;58(1):55-58.
127. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry. 1976;72:248-254.
128. 張惠婷. 順氯氨鉑引發的腎毒性在純系小鼠的確立與柴胡在此腎炎模型的藥效評估. 台北市: 藥學系碩士班, 臺北醫學大學; 2005.
129. 邱芷瑩. 人參與人參皂苷在順氯氨鉑引發的腎毒性於純系小鼠的藥效評估. 台北市: 藥學系碩士班, 臺北醫學大學; 2007.
130. Hultberg B, Ravnskov U. The excretion of N-acetyl-beta-glucosaminidase in glomerulonephritis. Clinical Nephrology. 1981;15(1):33-38.
131. Yokozawa T, Zhou JJ, Hattori M, Inaba S, Okada T, Oura H. Effects of ginseng in nephrectomized rats. Biological & Pharmaceutical Bulletin. 1994;17(11):1485-1489.
132. Han SW, Kim H. Ginsenosides stimulate endogenous production of nitric oxide in rat kidney. International Journal of Biochemistry & Cell Biology. May 1996;28(5):573-580.
133. Hattori T, Ito M, Suzuki Y, Hattori T, Ito M, Suzuki Y. Studies on antinephritic effects of plant components in rats (2): Effects of ginsenosides on original-type anti-GBM nephritis in rats and its mechanisms. Nippon Yakurigaku Zasshi - Folia Pharmacologica Japonica. 1991;97(2):127-134.
134. Wang Y, Wang BX, Liu TH, Minami M, Nagata T, Ikejima T. Metabolism of ginsenoside Rg1 by intestinal bacteria. II. Immunological activity of ginsenoside Rg1 and Rh1. Acta Pharmacol Sin. 2000;21(9):792-796.
135. Cho JY, Yoo ES, Baik KU, Park MH, Han BH. In vitro inhibitory effect of protopanaxadiol ginsenosides on tumor necrosis factor (TNF)-alpha production and its modulation by known TNF-alpha antagonists. Planta Med. 2001;67(3):213-218.
136. Park EK, Choo MK, Han MJ, Kim DH. Ginsenoside Rh1 possesses antiallergic and anti-inflammatory activities. Int Arch Allergy Immunol. 2004;133(2):113-120.
137. Shin YW, Bae EA, Kim SS, Lee YC, Kim DH. Effect of ginsenoside Rb1 and compound K in chronic oxazolone-induced mouse dermatitis. Int Immunopharmacol. 2005;5(7-8):1183-1191.
138. Park EK, Shin YW, Lee HU, Kim SS, Lee YC, Lee BY, Kim DH. Inhibitory effect of ginsenoside Rb1 and compound K on NO and prostaglandin E2 biosyntheses of RAW264.7 cells induced by lipopolysaccharide. Biol Pharm Bull. 2005;28(4):652-656.
139. Chen SM, Sato N, Yoshida M, Satoh N, Ueda S. Effects of Bupleurum scorzoneraefolium, Bupleurum falcatum, and saponins on nephrotoxic serum nephritis in mice. J Ethnopharmacol. 2008;116(3):397-402.
140. Leung KW, Leung FP, Huang Y, Mak NK, Wong RN. Non-genomic effects of ginsenoside-Re in endothelial cells via glucocorticoid receptor. FEBS Lett. 2007;581(13):2423-2428.
141. Yang CS, Ko SR, Cho BG,Shin D, Yuk JM, Li S, Kim JM, Evans RM, Jung JS, Song DK, Jo EK. The Ginsenoside Metabolite Compound K, a Novel Agonist of Glucocorticoid Receptor, Induces Tolerance to Endotoxin-induced Lethal Shock. J Cell Mol Med. 2007.
142. Dickey DT, Wu YJ, Muldoon LL, Neuwelt EA. Protection against cisplatin-induced toxicities by N-acetylcysteine and sodium thiosulfate as assessed at the molecular, cellular, and in vivo levels. Journal of Pharmacology & Experimental Therapeutics. 2005;314(3):1052-1058.
143. Luo J, Tsuji T, Yasuda H, Sun Y, Fujigaki Y, Hishida A. The molecular mechanisms of the attenuation of cisplatin-induced acute renal failure by N-acetylcysteine in rats. Nephrol Dial Transplant. 2008.
144. Anderson ME, Naganuma A, Meister A. Protection against cisplatin toxicity by administration of glutathione ester. FASEB J. 1990;4(14):3251-3255.
145. Wu YJ, Muldoon LL, Neuwelt EA. The chemoprotective agent N-acetylcysteine blocks cisplatin-induced apoptosis through caspase signaling pathway. Journal of Pharmacology & Experimental Therapeutics. 2005;312(2):424-431.
146. Sekharam M, Trotti A, Cunnick JM, Wu J. Suppression of fibroblast cell cycle progression in G1 phase by N-acetylcysteine. Toxicol Appl Pharmacol. 1998;149(2):210-216.
147. Doolan PD, Alpen EL, Theil GB. A clinical appraisal of the plasma concentration and endogenous clearance of creatinine. Am J Med. 1962;32:65-79.

 


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