進階搜尋


  查詢北醫館藏
系統識別號 U0007-3107201117033900
論文名稱(中文) 併用Doxorubicin或Paclitaxel與反譯寡核苷酸微脂粒對白血病細胞之療效探討
論文名稱(英文) The Combined Anticancer Effect of Doxorubicin or Paclitaxel with Liposomal Antisense Oligodeoxynucleotidesin Leukemia Cell Lines.
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 100
研究生(中文) 陳宗伯
研究生(英文) Zhong-Bo Chen
學號 M301098016
學位類別 碩士
語文別 中文
口試日期 2011-06-29
論文頁數 95頁
口試委員 指導教授-邱士娟
委員-胡德民
委員-陳建中
中文關鍵字 反譯寡核苷    微脂粒  Bcl-2  G3139 
英文關鍵字 antisense oligodeoxynuclotides  liposome  Bcl-2  G3139 
學科別分類
中文摘要 白血病為國內外的高發性惡性腫瘤之一,可分為急性、慢性的淋巴球白血病與骨髓性白血病。癌症的發生可能和癌細胞之程式性死亡-細胞凋亡(apoptosis)無法正常進行有關。細胞凋亡又稱為細胞程式死亡,為一受到許多因子調節之複雜過程,其目的為移除生物體所不需要或是已失去生理活性之細胞。Bcl-2蛋白質家族為調控細胞凋亡的因子之一,其中的Bcl-2蛋白之作用為抑制細胞凋亡,許多癌細胞Bcl-2基因過度表現,細胞凋亡無法正常發生,導致癌細胞過度增生,如果能夠降低癌細胞之Bcl-2蛋白質的表現,即可誘導癌細胞之細胞凋亡發生。
反譯治療(antisense therapy)是將特定基因材料,如反譯寡核苷酸(oligodeoxynucleotides, ODN)導入細胞以減少其不正常基因之表現。本實驗所使用之反譯寡核苷酸為G3139,是一種對Bcl-2 mRNA有專一性的反譯寡核苷酸,使細胞Bcl-2蛋白質的表現減少。
 本實驗之目的為藉由利用G3139微脂粒以增加化療藥物:doxorubicin或paclitaxel之抗癌效果。實驗中所使用的微脂粒由DC-Chol/egg-PC/PEG-DSPE 以22.5:76:1.5 mol%與G3139所組成,本實驗所使用之細胞株包含:K562、NCI-H929及CCRF-CEM。在細胞攝入的實驗部分,利用流式細胞儀以及螢光顯微鏡來觀察及定量細胞對於G3139微脂粒之攝入能力。同時利用西方墨點法來測定Bcl-2的蛋白質表現量。最後觀察在給藥之後24及48小時之細胞存活率,檢視藥物之細胞毒殺能力。
G3139微脂粒之粒徑大小平均為148.9±2.5 nm;在4℃之環境下儲存,在七週內其粒徑沒有明顯之改變;包覆ODN 的能力約為85.5±5.3%,表面電荷為17.5±1.3mV。實驗結果顯示,隨著投與之G3139濃度提升細胞之螢光表現也較強,表示G3139可以有效地將被導入細胞中,然而和未被包覆的G3139相比,經微脂粒乘載之G3139並沒有顯著增加G3139之攝入;同時在給予1 μM G3139微脂粒24小時之後可以發現Bcl-2蛋白質之表現有些許之下降。最後在細胞存活性試驗觀察到不論是在24小時或是48小時之組別中,高濃度的G3139微脂粒皆有較強的細胞毒殺能力。不同之細胞株對於化療藥物之敏感性也有不同,其中K562對於此兩種化療藥物皆有著較強的敏感性;而併用G3139微脂粒之後可增加paclitaxel對於NCI-H929之毒殺能力,但對於K562則無太大影響。此結果可能和細胞本身之敏感性有關,未來可以繼續朝著不同藥物對於不同細胞株之不同毒殺能力繼續探討。
英文摘要 Leukemia is one of the high incidence domestic malignancies. Leukemia can be divided into acute and chronic lymphocytic leukemia and acute and chronic myelogenous leukemia. Cancer may arise from the dysfunction in the apoptotic pathway. Apoptosis, or programmed cell death, is a highly regulated process that allows a cell to self-degrade in order for the body to eliminate unwanted or dysfunctional cells. One of the apoptosis-regulator is Bcl-2 family and Bcl-2, a member of this family, is an important anti-apoptotic protein in regulating the apoptosis pathway. Downregulation of Bcl-2 may be able to induce apoptosis, and further increase cell death in tumor cells.
Antisense therapy is a strategy of anticancer therapy designed to introduce genetic material, antisense oligodeoxynuclotides (ODNs), into cells to downregulate abnormal genes. G3139, an antisense ODN designed to specifically bind to Bcl-2 mRNA and further downregulate Bcl-2 protein expression, was used in this study.
The aim of this study was to enhance the anticancer effect of chemotherapeutic agents, Doxorubicin or Paclitaxel, using G3139-containing liposomes. The liposomes are composed of DC-Chol / egg-PC/ PEG-DSPE (22.5:76:1.5 mol%) and G3139. Leukemia-related cell lines, K562, NCI-H929 and CCRF-CEM, were used in this study. Cellular uptake of different formulations of liposomal G3139 was observed by flow cytometer and fluorescent microscope. In the meantime, the Bcl-2 protein level was evaluated by western blot to confirm whether G3139 work or not in selective cancer cells. Cell viability was evaluated by MTS assay in 24 and 48hr.
G3139-containing liposomes had a mean diameter of 148.9±2.5 nm. No significant particle size changes were observed for 7 weeks at 4℃. Encapsulation efficiency of ODNs in the liposomes was 85.5±5.3% and the zeta-potential was 17.5±1.3mV.The results in cellular uptake studies demonstrated that G3139 can be delivered into cells as higher concentration of G3139-liposomes exhibited higher fluorescence intensity. However, no significant difference was observed between free G3139 and G3139-liposomes. Slightly higher Bcl-2 downregulation effect was observed in cells treated with 1 ?嵱 G3139-liposomes at 24 hr. In cytotoxicity studies, cells treated with higher concentration of G3139 showed higher cytoxiticity in both time points. Three cell lines used in the current study exhibited different sensitivity to chemotherapeutic agents, as K562 had lowest IC50 in both drugs.G3139-liposomes could enhance the cytotoxic effect to paclitaxel in NCI-H929 cells but not in K562 cells. This may be due to the difference in sensitivity of chemotherapeutic agents in these cell lines. Further studies are needed to confirm this effect.
論文目次 中文摘要 I
英文摘要 III
目錄 V
圖目錄 VII
表目錄 IX
縮寫表 X
緒論 1
白血病簡介 1
白血病的治療 5
基因治療與反譯治療 6
細胞凋亡 8
Bcl-2蛋白家族 11
反譯寡核苷酸 13
G3139(Oblimersen, Genasense™) 14
基因運輸系統 16
微脂粒 19
化學藥物治療 22
Doxorubicin 24
Paclitaxel 26
研究動機與目的 18
實驗材料與方法 29
實驗材料 29
實驗細胞 29
實驗藥品 30
實驗儀器 32
實驗方法 33
細胞培養 33
微脂粒製備 35
微脂粒包覆能力試驗 36
細胞攝入試驗 37
基因轉染試驗 39
細胞蛋白質萃取與定量 40
西方墨點法分析Bcl-2蛋白質表現 41
細胞毒殺性試驗 44
統計方法 47
實驗結果 48
微脂粒之物理化學性質特性 48
微脂粒之表面電荷、型態及粒徑 49
微脂粒包覆反譯寡核苷酸能力 49
微脂粒膠體安定性試驗 50
微脂粒之體外試驗 52
細胞攝入試驗 52
流式細胞儀 52
螢光顯微鏡 59
基因轉染試驗 63
反譯寡核苷酸與化療藥物之細胞存活率試驗 66
討論 84
結論 88
參考文獻 89
參考文獻 1. Pooley, R.J., Clinical hematology atlas. Arch Pathol Lab Med, 1999. 123(11): p. 1125.
2. Henderson, L.M. and J.B. Chappel, NADPH oxidase of neutrophils. Biochim Biophys Acta, 1996. 1273(2): p. 87-107.
3. Kakizuka, A., et al., Chromosomal translocation t(15;17) in human acute promyelocytic leukemia fuses RAR alpha with a novel putative transcription factor, PML. Cell, 1991. 66(4): p. 663-74.
4. Landis, S.H., et al., Cancer statistics, 1998. CA Cancer J Clin, 1998. 48(1): p. 6-29.
5. Stone, R.M. and R.J. Mayer, Treatment of the newly diagnosed adult with de novo acute myeloid leukemia. Hematol Oncol Clin North Am, 1993. 7(1): p. 47-64.
6. Hoelzer, D.F., Therapy of the newly diagnosed adult with acute lymphoblastic leukemia. Hematol Oncol Clin North Am, 1993. 7(1): p. 139-60.
7. Kantarjian, H.M., et al., Prolonged survival in chronic myelogenous leukemia after cytogenetic response to interferon-alpha therapy. The Leukemia Service. Ann Intern Med, 1995. 122(4): p. 254-61.
8. Faguet, G.B., Chronic lymphocytic leukemia: an updated review. J Clin Oncol, 1994. 12(9): p. 1974-90.
9. Kalyn, R., Overview of targeted therapies in oncology. J Oncol Pharm Pract, 2007. 13(4): p. 199-205.
10. Crooke, S.T., Potential roles of antisense technology in cancer chemotherapy. Oncogene, 2000. 19(56): p. 6651-9.
11. Milhavet, O., D.S. Gary, and M.P. Mattson, RNA interference in biology and medicine. Pharmacol Rev, 2003. 55(4): p. 629-48.
12. Morille, M., et al., Progress in developing cationic vectors for non-viral systemic gene therapy against cancer. Biomaterials, 2008. 29(24-25): p. 3477-96.
13. Rayburn, E.R. and R. Zhang, Antisense, RNAi, and gene silencing strategies for therapy: mission possible or impossible? Drug Discov Today, 2008. 13(11-12): p. 513-21.
14. Yao, Y., et al., Antisense makes sense in engineered regenerative medicine. Pharm Res, 2009. 26(2): p. 263-75.
15. Gary, D.J., N. Puri, and Y.Y. Won, Polymer-based siRNA delivery: perspectives on the fundamental and phenomenological distinctions from polymer-based DNA delivery. J Control Release, 2007. 121(1-2): p. 64-73.
16. Kerr, J.F., A.H. Wyllie, and A.R. Currie, Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer, 1972. 26(4): p. 239-57.
17. Lockshin, R.A. and J. Beaulaton, Programmed cell death. Life Sci, 1974. 15(9): p. 1549-65.
18. Zakeri, Z. and R.A. Lockshin, Physiological cell death during development and its relationship to aging. Ann N Y Acad Sci, 1994. 719: p. 212-29.
19. Ishizaki, Y., [Physiological functions of programmed cell death]. Seikagaku, 1998. 70(5): p. 365-70.
20. Reed, J.C., Mechanisms of apoptosis avoidance in cancer. Curr Opin Oncol, 1999. 11(1): p. 68-75.
21. Behl, C., Apoptosis and Alzheimer's disease. J Neural Transm, 2000. 107(11): p. 1325-44.
22. Cotman, C.W., et al., Possible role of apoptosis in Alzheimer's disease. Ann N Y Acad Sci, 1994. 747: p. 36-49.
23. Searle, J., J.F. Kerr, and C.J. Bishop, Necrosis and apoptosis: distinct modes of cell death with fundamentally different significance. Pathol Annu, 1982. 17 Pt 2: p. 229-59.
24. Matsuda, H., et al., Apoptosis and necrosis occurring during different stages of primary and metastatic tumor growth of a rat mammary adenocarcinoma. Anticancer Res, 1996. 16(3A): p. 1117-21.
25. Nicotera, P., M. Leist, and E. Ferrando-May, Apoptosis and necrosis: different execution of the same death. Biochem Soc Symp, 1999. 66: p. 69-73.
26. Li, H., H. Sun, and Z.M. Qian, The role of the transferrin-transferrin-receptor system in drug delivery and targeting. Trends Pharmacol Sci, 2002. 23(5): p. 206-9.
27. Danial, N.N., BCL-2 family proteins: critical checkpoints of apoptotic cell death. Clin Cancer Res, 2007. 13(24): p. 7254-63.
28. Marzo, I. and J. Naval, Bcl-2 family members as molecular targets in cancer therapy. Biochem Pharmacol, 2008. 76(8): p. 939-46.
29. Tsujimoto, Y. and S. Shimizu, Bcl-2 family: life-or-death switch. FEBS Lett, 2000. 466(1): p. 6-10.
30. Cimmino, A., et al., miR-15 and miR-16 induce apoptosis by targeting BCL2. Proc Natl Acad Sci U S A, 2005. 102(39): p. 13944-9.
31. Campos, L., et al., High expression of bcl-2 protein in acute myeloid leukemia cells is associated with poor response to chemotherapy. Blood, 1993. 81(11): p. 3091-6.
32. Segal, N.H., et al., BCL-2 proto-oncogene expression in prostate cancer and its relationship to the prostatic neuroendocrine cell. Arch Pathol Lab Med, 1994. 118(6): p. 616-8.
33. Webb, A., et al., BCL-2 antisense therapy in patients with non-Hodgkin lymphoma. Lancet, 1997. 349(9059): p. 1137-41.
34. De Rosa, G., et al., Long-term release and improved intracellular penetration of oligonucleotide-polyethylenimine complexes entrapped in biodegradable microspheres. Biomacromolecules, 2003. 4(3): p. 529-36.
35. Pan, X., et al., Antitumor activity of G3139 lipid nanoparticles (LNPs). Mol Pharm, 2009. 6(1): p. 211-20.
36. Dias, N. and C.A. Stein, Potential roles of antisense oligonucleotides in cancer therapy. The example of Bcl-2 antisense oligonucleotides. Eur J Pharm Biopharm, 2002. 54(3): p. 263-9.
37. Moulder, S.L., et al., Phase I/II study of G3139 (Bcl-2 antisense oligonucleotide) in combination with doxorubicin and docetaxel in breast cancer. Clin Cancer Res, 2008. 14(23): p. 7909-16.
38. Lai, J.C., et al., A pharmacologic target of G3139 in melanoma cells may be the mitochondrial VDAC. Proc Natl Acad Sci U S A, 2006. 103(19): p. 7494-9.
39. Rudin, C.M., et al., Phase I study of G3139, a bcl-2 antisense oligonucleotide, combined with carboplatin and etoposide in patients with small-cell lung cancer. J Clin Oncol, 2004. 22(6): p. 1110-7.
40. Advani, P.P., et al., Pharmacokinetic evaluation of oblimersen sodium for the treatment of chronic lymphocytic leukemia. Expert Opin Drug Metab Toxicol, 2011. 7(6): p. 765-74.
41. Abou-Nassar, K. and J.R. Brown, Novel agents for the treatment of chronic lymphocytic leukemia. Clin Adv Hematol Oncol, 2010. 8(12): p. 886-95.
42. Loriot, Y., et al., Inhibition of BCL-2 in small cell lung cancer cell lines with oblimersen, an antisense BCL-2 oligodeoxynucleotide (ODN): in vitro and in vivo enhancement of radiation response. Anticancer Res, 2010. 30(10): p. 3869-78.
43. Klasa, R.J., et al., Oblimersen Bcl-2 antisense: facilitating apoptosis in anticancer treatment. Antisense Nucleic Acid Drug Dev, 2002. 12(3): p. 193-213.
44. Guowei Dai, M.S., PHARMACOKINETICS, PHARMACODYNAMICS AND METABOLISM OF BCL-2 ANTISENSE PHOSPHOROTHIOATE OLIGONUCLEOTIDE G3139 (GENASENSE®). 2005.
45. Oblimersen: Augmerosen, BCL-2 antisense oligonucleotide - Genta, G 3139, GC 3139, oblimersen sodium. Drugs R D, 2007. 8(5): p. 321-34.
46. Wolff, J.A., et al., Direct gene transfer into mouse muscle in vivo. Science, 1990. 247(4949 Pt 1): p. 1465-8.
47. Gao, X., K.S. Kim, and D. Liu, Nonviral gene delivery: what we know and what is next. AAPS J, 2007. 9(1): p. E92-104.
48. Li, S.D. and L. Huang, Non-viral is superior to viral gene delivery. J Control Release, 2007. 123(3): p. 181-3.
49. Walther, W. and U. Stein, Viral vectors for gene transfer: a review of their use in the treatment of human diseases. Drugs, 2000. 60(2): p. 249-71.
50. Al-Dosari, M.S. and X. Gao, Nonviral gene delivery: principle, limitations, and recent progress. AAPS J, 2009. 11(4): p. 671-81.
51. Felgner, P.L., et al., Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci U S A, 1987. 84(21): p. 7413-7.
52. Tranchant, I., et al., Physicochemical optimisation of plasmid delivery by cationic lipids. J Gene Med, 2004. 6 Suppl 1: p. S24-35.
53. Dass, C.R. and P.F. Choong, Targeting of small molecule anticancer drugs to the tumour and its vasculature using cationic liposomes: lessons from gene therapy. Cancer Cell Int, 2006. 6: p. 17.
54. Dickson, D., UK scientists test liposome gene therapy technique. Nature, 1993. 365(6441): p. 4.
55. Bangham, A.D., M.M. Standish, and J.C. Watkins, Diffusion of univalent ions across the lamellae of swollen phospholipids. J Mol Biol, 1965. 13(1): p. 238-52.
56. Frezard, F. and A. Garnier-Suillerot, Permeability of lipid bilayer to anthracycline derivatives. Role of the bilayer composition and of the temperature. Biochim Biophys Acta, 1998. 1389(1): p. 13-22.
57. Gregoriadis, G., The carrier potential of liposomes in biology and medicine (first of two parts). N Engl J Med, 1976. 295(13): p. 704-10.
58. Farhood, H., N. Serbina, and L. Huang, The role of dioleoyl phosphatidylethanolamine in cationic liposome mediated gene transfer. Biochim Biophys Acta, 1995. 1235(2): p. 289-95.
59. Long term trends in cancer mortality rates from 1955 to 1987 in Japan. The Bureau of Vital Statistics, Ministry of Health and Welfare. Jpn J Clin Oncol, 1989. 19(3): p. 305-17.
60. Bhattacharya, S. and S. Haldar, Interactions between cholesterol and lipids in bilayer membranes. Role of lipid headgroup and hydrocarbon chain-backbone linkage. Biochim Biophys Acta, 2000. 1467(1): p. 39-53.
61. Dunn, S.E., et al., Polystyrene-poly (ethylene glycol) (PS-PEG2000) particles as model systems for site specific drug delivery. 2. The effect of PEG surface density on the in vitro cell interaction and in vivo biodistribution. Pharm Res, 1994. 11(7): p. 1016-22.
62. Gabizon, A.A., Y. Barenholz, and M. Bialer, Prolongation of the circulation time of doxorubicin encapsulated in liposomes containing a polyethylene glycol-derivatized phospholipid: pharmacokinetic studies in rodents and dogs. Pharm Res, 1993. 10(5): p. 703-8.
63. Karnofsky, D.A., I. Graef, and H.W. Smith, Studies on the mechanism of action of the nitrogen and sulfur mustards in vivo. Am J Pathol, 1948. 24(2): p. 275-91.
64. Farber, S. and L.K. Diamond, Temporary remissions in acute leukemia in children produced by folic acid antagonist, 4-aminopteroyl-glutamic acid. N Engl J Med, 1948. 238(23): p. 787-93.
65. Zwelling, L.A., et al., Protein-associated deoxyribonucleic acid strand breaks in L1210 cells treated with the deoxyribonucleic acid intercalating agents 4'-(9-acridinylamino) methanesulfon-m-anisidide and adriamycin. Biochemistry, 1981. 20(23): p. 6553-63.
66. Nelson, E.M., K.M. Tewey, and L.F. Liu, Mechanism of antitumor drug action: poisoning of mammalian DNA topoisomerase II on DNA by 4'-(9-acridinylamino)-methanesulfon-m-anisidide. Proc Natl Acad Sci U S A, 1984. 81(5): p. 1361-5.
67. Gorodetsky, R., et al., Liposome transduction into cells enhanced by haptotactic peptides (Haptides) homologous to fibrinogen C-termini. J Control Release, 2004. 95(3): p. 477-88.
68. Gabizon, A., R. Shiota, and D. Papahadjopoulos, Pharmacokinetics and tissue distribution of doxorubicin encapsulated in stable liposomes with long circulation times. J Natl Cancer Inst, 1989. 81(19): p. 1484-8.
69. Eliaz, R.E. and F.C. Szoka, Jr., Liposome-encapsulated doxorubicin targeted to CD44: a strategy to kill CD44-overexpressing tumor cells. Cancer Res, 2001. 61(6): p. 2592-601.
70. Batist, G., et al., Reduced cardiotoxicity and preserved antitumor efficacy of liposome-encapsulated doxorubicin and cyclophosphamide compared with conventional doxorubicin and cyclophosphamide in a randomized, multicenter trial of metastatic breast cancer. J Clin Oncol, 2001. 19(5): p. 1444-54.
71. Shapiro, C.L., et al., Phase II trial of high-dose liposome-encapsulated doxorubicin with granulocyte colony-stimulating factor in metastatic breast cancer. TLC D-99 Study Group. J Clin Oncol, 1999. 17(5): p. 1435-41.
72. Simoes-Wust, A.P., et al., Bcl-2/bcl-xL bispecific antisense treatment sensitizes breast carcinoma cells to doxorubicin, paclitaxel and cyclophosphamide. Breast Cancer Res Treat, 2002. 76(2): p. 157-66.
73. Schondorf, T., et al., Interaction of cisplatin, paclitaxel and adriamycin with the tumor suppressor PTEN. Anticancer Drugs, 2001. 12(10): p. 797-800.
74. Rosenblum, M.D. and R.R. Shivers, 'Rings' of F-actin form around the nucleus in cultured human MCF7 adenocarcinoma cells upon exposure to both taxol and taxotere. Comp Biochem Physiol C Toxicol Pharmacol, 2000. 125(1): p. 121-31.
75. Chiu, S.J., et al., Efficient delivery of a Bcl-2-specific antisense oligodeoxyribonucleotide (G3139) via transferrin receptor-targeted liposomes. J Control Release, 2006. 112(2): p. 199-207.
76. Meure, L.A., N.R. Foster, and F. Dehghani, Conventional and dense gas techniques for the production of liposomes: a review. AAPS PharmSciTech, 2008. 9(3): p. 798-809.
77. Ulrich, A.S., Biophysical aspects of using liposomes as delivery vehicles. Biosci Rep, 2002. 22(2): p. 129-50.
78. 成恆宇, 利用反譯寡核苷酸微脂粒-G3139提升Paclitaxel或Doxorubicin於癌細胞之敏感性. 2010.


論文全文使用權限
  • 同意授權瀏覽/列印電子全文服務,於2016-08-18起公開。


  • 若您有任何疑問,請與我們聯絡!
    臺北醫學大學 圖書館 簡莉婷
    E-mail:etds@tmu.edu.tw
    Tel:(02) 2736-1661 ext.2519
    Fax:(02) 2737-5446