進階搜尋


  查詢北醫館藏
系統識別號 U0007-1607201015575000
論文名稱(中文) 白藜蘆醇對高糖引起小老鼠間質細胞生長抑制現象的作用
論文名稱(英文) The Effects of Resveratrol on High Glucose-Induced Growth Arrest in Mouse Mesangial Cells
校院名稱 臺北醫學大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Graduate Institute of Clinical Medicine
學年度 98
學期 2
出版年 99
研究生(中文) 陳怡安
研究生(英文) Yi-Ann Chen
學號 M118097002
學位類別 碩士
語文別 中文
口試日期 2010-06-10
論文頁數 72頁
口試委員 指導教授-林裕峰
委員-簡志誠
委員-許永和
中文關鍵字 白藜蘆醇  高糖環境  抗氧化  凋亡 
英文關鍵字 Resveratrol  high glucose  antioxidant  apoptosis 
學科別分類
中文摘要 背景:糖尿病腎病變是造成腎衰竭最主要的原因之一,血糖的控制不佳會增加心血管疾病的發生率。慢性的高糖環境下會對腎臟細胞,形成莫大的氧化壓力,氧化產物的過度製造增加,導致腎臟間質細胞的生長抑制及凋亡。我們評估白藜蘆醇這一個多酚構造、多功能的植物天然抗毒素是否可經由抗氧化、抗發炎的過程,減少小鼠腎臟間質細胞暴露在高糖環境下生長抑制及凋亡的現象。研究方法與材料:小鼠腎臟間質細胞分別被培養在含有6.25mM 較低糖分,及35mM 較高糖分環境下,和加入白藜蘆醇做治療。藉由顯微鏡、MTT test觀察其生長狀況。H2-DCFDA偵測細胞內過氧化物質含量。細胞的凋亡判定是運用流式細胞儀和Annexin-V以及propidium iodine的染色。藉由反轉錄聚合脢連鎖反應實驗測量TGFβ及HO-1含量。以及使用ELISA kit 定量 caspsae3。結果:高糖環境會有抑制間質細胞生長、造成細胞內ROS 上升(約增加~70 %;p < 0.05)及促進細胞凋亡現象(增加約1.5~1.8倍;p < 0.05) 。這些反應可因加入合理的生理劑量內的白藜蘆醇(1-2.5μM)而減少。此外也可以下降TGF-β,誘導HO-1酵素生成。可阻斷caspase 3 活性在高糖環境下(p < 0.05)。結論:我們證實了白藜蘆醇經由抗氧化過程可以減緩小鼠腎臟間質細胞暴露在高糖環境下凋亡的現象,而可以改善間質細胞數目的平衡。
英文摘要 Background: Diabetic nephropathy is one major cause of renal failure, and poor glycemic control will increase cardiovascular incidence. Chronic high glucose condition induced oxidative stress, increased reactive oxygen species (ROS) amount, and involves kidney mesangial cell growth arrest and apoptosis pathway. We investigated the therapeutic effect of resveratrol, a polyphenolic compound, with multi-function nature phytoalexin may attenuate high glucose induced cell arrest and apoptosis changes in mouse mesangial cell via anti-oxidant and anti-inflammation proprieties. Material and methods: Mouse mesangial cell were cultured in 6.25mM and 35mM glucose, and add resveratrol in high glucose treated group. Observe cell morphology and viability by microscopy and MTT test. Intracellular ROS generation was assayed by H2-DCFDA. Cell apoptosis amount was determined by flow cytometry via Annexin-V and propidium iodine staining. TGF and HO-1 gene expression was measured by reveres transcriptase- polymerase chain reaction. And caspase 3 activity was quantified by ELISA. Result: High glucose depressed cell growth, increases cellular ROS (~70 %; p < 0.05), and enhanced cell apoptosis (1.5~1.8 fold increase; p < 0.05); these reaction could be attenuated by physiological resveratrol concentration 1-2.5μM. And also noted decreased TGF-β and mediated HO-1 gene expression. Caspase 3 activity was also blocked (p < 0.05). Conclusion: Our findings indicate that resveratrol provides antioxidant effect against high glucose induced cell apoptosis, and may further regulate mesangial cell numbers.
論文目次 中文摘要 (Abstract in Chinese) ----------------------- i
英文摘要 (Abstract in English) ----------------------- ii
第一章 緒論 (Introduction) -----------------------1
第一節 腎臟的構造及糖尿病腎病變的形成--------------- 4
第二節 高糖(High glucose)造成細胞的凋亡( apoptosis)及死亡(death ) -----------------------5
第三節 Transforming growth factor-beta(TGF-β)----------------------- 8
第四節 血红素氧合酶1 (HO-1) ----------------------- 10
第五節 白藜蘆醇(Resveratrol)的藥理作用及臨床應----------------------12
第六節 抗氧化物在腎臟的運用----------------------- 15
第七節 研究目的 ----------------------- 16
第二章 研究方法與材料 (Materials and Methods) ----------------------- 17
第三章 研究結果 (Results)
1.Effect of high glucose on mouse mesangial cell viability ------------------- 26
2.Effects of different dosage resveratrol on mouse mesangial cell viability -----------------------27
3.Effect of resveratrol on high glucose-induced oxidative stress in mouse mesangial cell -----------------------28
4.Effect of resveratrol on high glucose-induced cell apoptosis in mouse mesangial cell-----------------------28
5.Effectof resveratrol on high glucose-induced TGF-β in mouse mesangial cell-----------------------29
6.Effect.of resveratrol mediated HO-1 expression on high glucose treatment in mouse mesangial cell-----------------------30
7.Effect of resveratrol on high glucose-induced caspase-3 activation in mouse mesangial cell-----------------------30
第四章 討論 (Discussion) -----------------------31
第五章 結論與展望 (Conclusion and Perspective) -----------------------45
參考文獻 (References) ----------------------- 46
圖 (Figures) ----------------------- 55
參考文獻 Ayo SH, Radnik RA, Glass WF, et al. Increased extracellular matrix synthesis and mRNA in mesangial cells grown in high glucose medium. Am J Physiol
1991;260:F185–F191.

Agarwal A, Balla J, Alam J ,et al. Induction of heme oxygenase in toxic
renal injury: a protective role in cisplatin nephrotoxicity in the rat. Kidney
Int 1995; 48: 1298–1307.

Abraham NG, Drummond GS, Lutton JD, et al. The biological significance
and physiological role of heme oxygenase. Cell Physiol Biochem 1996; 6:
129–168.

Aizawa T, Ishizaka N, Taguchi J ,et al. Heme oxygenase-1 is upregulated in the kidney of angiotensin II-induced hypertensive rats: possible role in renoprotection. Hypertension 2000; 35: 800–806.

Avihingsanon Y, Ma N, Csizmadia E,et al. Expression of protective genes in human renal allografts: a regulatory response to injury associated with graft rejection. Transplantation 2002; 73: 1079–1085.

Allen DA, Yaqoob MM, Harwood SM: Mechanisms of high glucose-induced apoptosis and its relationship to diabetic complications.J Nutr Biochem 2005; 16: 705–713.

Aziz MH., Nihal M., Fu VX., et al: Resveratrol-caused apoptosis of human prostate carcinoma LNCaP cells is mediated via modulation of phosphatidylinositol 3-kinase/Akt pathway and Bcl-2 family proteins. Mol. Cancer Ther.2006; 5: 1335–1341

Bertelli A, Bertelli AA,Gozzini A, et al. Plasma and tissue resveratrol concentrations and pharmacological activity. Drugs Exp Clin Res 1998; 24:133-138.

Bertelli AA, Ferrara F, Diana G, et al. Resveratrol, a natural stilbene in grapes and wine, enhances intraphagocytosis in human promonocytes: a co-factor in antiinflammatory and anticancer chemopreventive activity. Int J Tissue React 1999;21:93–104.

Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature 2001; 414:813– 20.

Bonnefont-Rousselot.Glucose and reactive oxygen species.Curr. Opin. Clin. Nutr. Metab. Care 2002;5: 561–568.

Bamri-Fzzine S, Zhu AO, Londono I ,et al. Apoptosis of tubular epithelial cells in glycogen nephrosis during diabetes. Lab Invest 2003 83: 1069–1080

Chase HP, Jackson WE, Hoops SL, et al. Glucose control and the renal and retinal complications of insulin dependant diabetes. J Am Med Assoc 1989;261: 1155–1160.

Chen S, Hong SW, Iglesias DL ,et al. The key role of the transforming growth factor-β system in the pathogenesis of diabetic nephropathy. Ren Fail 2001; 23: 471–481

Catherwood MA, Powell LA, Anderson P, et al. Glucose-induced oxidative stress in mesangial cells.Kidney Int 2002;61:599– 608.

Cichewicz RH, Kouzi SA.Resveratrol oligomers: structure, chemistry and biological activity, in: A.U. Rahman (Ed.), Studies in Natural Products Chemistry,vol. 26, Elsevier BV, Oxford, UK, 2002, pp. 507–579.

Chen S, Hoffman BB, Lee J, et al. Cultured tubule cells from TGF-β1 null mice exhibit impaired hypertrophy and fibronectin expression in high glucose .Kidney Int 2004; 65: 1191–1204.

Cohen, H. Y., Miller, C., Bitterman, K. J,et al. Calorie restriction promotes mammalian cell survival by inducing the SIRT1 deacetylase. Science. 2004;305:
390–392.

Crowell JA, Korytko PJ, Morrissey RL,et al. Resveratrol-Associated Renal Toxicity. Toxico. Sciences 2004;82: 614–619.

Chan WH. Effect of Resveratrol on High Glucose-Induced Stress in Human Leukemia K562 Cells. J. Cell. Biochem. 2005; 94: 1267–1279..

Das DK, Sato M, Ray PS, et al. Cardioprotection of red wine: role of polyphenolic antioxidants. Drugs Under Experimental and Clinical Research 1999;25: 115– 120
Dworkin LD, Ichikawa I, Brenner BM. Hormonal modulation of glomerular function. Am J Physiol 1983;244:F95–F104.

Frankel EN, Waterhouse AL, Kinsella JE. Inhibition of human LDL oxidation by resveratrol. Lancet 1993; 341:1103–1104.

Fremont L. Biological effects of resveratrol. Life Sci 2000;66: 663–73.

Friedlander RM: Apoptosis and caspases in neurodegenerative diseases. N Engl J Med 2003; 348: 1365–1375.

Fulda, S., and Debatin, K. M. Resveratrol modulation of signal transduction in apoptosis and cell survival: a mini-review. Cancer Detect. Prev. 2006; 30: 217–223.

Green DR, Reed JC: Mitochondria and apoptosis. Science 1998; 281: 1309–1312.

Gagliardini G, Benigni A.Role of anti-TGF-β antibodies in the treatment of renal injury Cytokines and Growth factor 2006;16: 89-96.

Goodman AI, Olszanecki R, Yang LM, et al. Heme oxygenase-1 protects against radiocontrast-induced acute kidney injury by regulating antiapoptotic proteins. Kidney Int 2007; 72: 945–953.

Henry DN, Busik JV, Brosius FC, et al. Glucose transporters control gene expression of aldose reductase, PKC and GLUT1 in mesangial cell in vitro. Am J Physiol 1999;277:F97–F104.

Haugen EN, Croatt AJ, Nath KA. Angiotensin II induces renal oxidant stress in vivo and heme oxygenase-1 in vivo and in vitro. Kidney Int 2000; 58: 144–152

Hill-Kapturczak N, Truong L, Thamilselvan V, et al. Smad7-dependent regulation of heme oxygenase-1 by transforming growth factor-b in human renal epithelial cells. J Biol Chem 2000; 275: 40904–40909.

Hong SW, Isono M, Chen S, et al. Increased glomerular and tubular expression of transforming growth factor-β1, its type II receptor, and activation of the Smad signaling pathway in the db/db mouse. Am J Pathol 2001; 158: 1653–1663

Howitz KT,Bitterman KJ,Cohen HY et al. Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature 2003; 425, 191–196.

Haber CA, Lam TK, Yu Z, et al. N-Acetylcysteine and taurine prevent hyperglycemia-induced insulin resistance in vivo: possible role of oxidative stress. Am J Physi Endocrinol Metab 2003;285:E744– 753.

Hou DX, Uto T, Tong X, et al. Involvement of reactive oxygen species-independent mitochondrial pathway in gossypolinduced apoptosis. Arch Biochem Biophys 2004; 428: 179–187

Hung KY, Liu SY, Kao SH, et al. N-acetylcysteine-mediated antioxidation prevents hyperglycemia-induced apoptosis and collagen synthesis in rat mesangial cells. Am J Nephrol. 2009;29(3):192-202.

Isomaa B, Almgren P, Tuomi T, Forsen B, Lahti K, Nissen M, et al. Cardiovascular morbidity and mortality associated with the metabolic syndrome. Diabetes Care 2001; 24:683– 9.

Jang M, Cai L, Udeani GO, et al. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science 1997; 275: 218–20.

Jang M, Pezzuto JM. Cancer chemopreventive activity of resveratrol. Drugs Exp Clin Res 1999; 25:65–77.

Juan ME, Vinardell MP, Planas JM. The Daily Oral Administration of High Doses of trans-Resveratrol to Rats for 28 Days Is Not Harmful. J. Nutr. 2002; 132: 257–260.

Kreisberg JI, Venkatachalam M, Troyer D. Contractile properties of cultured glomerular mesangial cells. Am J Physiol 1985; 249(4 Part 2):F457–F463.

Kulkarni AB, Huh CG., Becker D, et al. Transforming growth factor-b1 null mutation in mice causes excessive inflammatory response and early death.
Proc. Natl. Acad. Sci. 1993; 90: 770–774..

Kanakiriya SK, Croatt AJ, Haggard JJ ,et al. Heme: a novel inducer of MCP-1 through HO-dependent and HO-independent mechanisms. Am J Physiol Renal Physiol 2003; 284: F546–F554.
Kang BP, Frencher S, Reddy V, et al.: High glucose promotes mesangial cell apoptosis by oxidant dependent mechanism. Am J Physiol Renal Physiol 2003; 284:F455–F466.

Kang BP, Urbonas A, Baddoo A, et al.: IGF-1 inhibits the mitochondrial apoptosis program in mesangial cells exposed to high glucose. Am J Physiol Renal Physiol 2003;285: F1013–F1024.

Ko CH, Shen SC, Hsu CS, et al.: Mitochondrial- dependent, reactive oxygen species-independent apoptosis by myricetin: roles of protein kinase C, cytochrome c, and caspase cascade. Biochem Pharmacol 2005; 69: 913–927

Khera T, Martin J, Riley S, et al. Glucose enhances mesangial cell apoptosis. Lab Invest 2006; 86: 566–577.

Kirkby KA, Adin CA. Products of heme oxygenase and their potential therapeutic applications. Am J Physiol Renal Physiol 2006; 290: F563–F571.

Kim OS, Kim YS, Jang DS, et al..Cytoprotection against hydrogen peroxide-induced cell death in cultured mouse mesangial cells by erigeroflavanone, a novel compound from the flowers of Erigeron annuus. Chem Biol Interact. 2009; 180:414-20.

Lemos FB, Ijzermans JN, Zondervan PE, et al. Differential expression of heme oxygenase-1 and vascular endothelial growth factor in cadaveric and living donor kidneys after ischemia–reperfusion. J Am Soc Nephrol 2003; 14: 3278–3287.

Lin CL, Wang JY, Huang YT, et al. Wnt/β-Catenin Signaling Modulates Survival of High Glucose–Stressed Mesangial Cells. J Am Soc Nephrol. 2006 ;17:2812-20

Maines MD, Veltman JC. Phenylhydrazine-mediated induction of haem oxygenase activity in rat liver and kidney and development of hyperbilirubinaemia. Inhibition by zinc-protoporphyrin. Biochem J 1984; 217: 409–417

Maines MD, Mayer RD, Ewing JF, et al. Induction of kidney heme oxygenase-1 (HSP32) mRNA and protein by ischemia/reperfusion: possible role of heme as both promotor of tissue damage and regulator of HSP32. J Pharmacol Exp Ther 1993; 264: 457–462

McLennan SV, Fisher EJ, Yue DK, et al. High glucose concentration causes a decrease in mesangium degradation. Diabetes 1994;43:1041–1046.

Mishra R, Emancipator SN, Kern T, et al. High glucose evokes an intrinsic proapoptotic signaling pathway in mesangial cells. Kidney Int 2005; 67:82–93

Mishra R, Emancipator SN, Kern T, et al. High glucose evokes an intrinsic proapoptotic signaling pathway in mesangial cells. Kidney Int 2005; 67:82–93.

Nishikawa T, Edelstein D, Du XL, et al. Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature 2000; 404: 787–790.

Morales AI, Rodrı´guez-Barbero A, Vicente-Sa´nchez C, et al. Resveratrol inhibits gentamicin-induced mesangial cell contraction. Life Sciences 2006;78: 2373 – 2377

Nicholson DW, Thornberry NA: Apoptosis. Life and death decisions. Science 2003; 299: 214–215

Nath KA. Heme oxygenase-1: a provenance for cytoprotective pathways in the kidney and other tissues. Kidney Int 2006; 70: 432–443

Odetti P, Pesce C, Traverso N, et al. Comparative trial of N-acetyl-cysteine, taurine, and oxerutin on skin and kidney damage in long-term experimental diabetes.
Diabetes 2003; 52:499–505.

Pugliese G, Pricci F, Mene P .High glucose level unmasks a genetic predisposition to enhanced extracellular matrix production in mesangial cells from the Milan normotensive strain. J Am Soc Nephrol 1997; l 8:406–414

Powell LA, Nally SM, McMaster D, etal. Restoration of glutathione levels in vascular smooth muscle cells exposed to high glucose conditions. Free Radic Biol Med 2001;
31:1149– 55.

Pesce C, Menini S, Pricci F, et al. Glomerular cell replication and cell loss through apoptosis in experimental diabetes mellitus. Nephron 2002; 90:484–488

Pedraza-Chaverri J, Murali NS, Croatt AJ,et al. Proteinuria as a determinant of renal expression of heme oxygenase-1: studies in models of glomerular and tubular proteinuria in the rat. Am J Physiol Renal Physiol 2006; 290: F196–F204.

Steffes MW, Osterby R, Chavers B, et al. Mesangial expansion as a central mechanism for loss of kidney function in diabetic patients. Diabetes 1989; 38: 1077–1081.

Sharmak K, Ziyadefh F. The emerging role of transforming growth factor-β in kidney diseases. Am J Physiol 1994; 35: F829–F842

Sharma K, Ziyadeh FN. Hyperglycaemia and diabetic kidney disease. Diabetes 1995;44:1139–1146.

Sharma K, Jin Y, Guo J ,et al. Neutralization of TGF-β by anti-TGF-β antibody attenuates kidney hypertrophy and the enhanced extracellular matrix gene expression in STZ-induced diabetic mice. Diabetes 1996; 45: 522–530

Slater I, Odum J, Ashby J. Resveratrol and red wine consumption. Hum Exp Toxicol 1999;18:625–6.

Shimizu H, Takahashi T, Suzuki T, et al. Protective effect of heme oxygenase induction in ischemic acute renal failure. Crit Care Med 2000; 28: 809–817.

Shiraishi F, Curtis LM, Truong L, et al. Heme oxygenase-1 gene ablation or expression modulates cisplatin-induced renal tubular apoptosis. Am J Physiol Renal Physiol 2000; 278: F726–F736

Sono M, Chen S, Hong SW, et al. Smad pathway is activated in the diabetic mouse kidney and Smad3 mediates TGF-β-induced fibronectin in mesangial cells. Biochem Biophys Res Commun 2002; 296: 1356–1365

Sinclair, D. A. Toward a unified theory of caloric restriction and longevity regulation. Mech. Ageing Dev. 2005; 126: 987–1002

Suenaga F, Hatsushika K, Takano S, et al: A possible link between resveratrol and TGF-β: Resveratrol induction of TGF-βexpression and signaling. FEBS Letters 2008; 582: 586-590

Tetsuka T, Daphna-Iken D, Srivastava SK, et al. Regulation of heme oxygenase mRNA in mesangial cells: prostaglandin E2 negatively modulates interleukin-1-induced heme oxygenase-1 mRNA. Biochem Biophys Res Commun 1995; 212: 617–623.

The diabetes control and complications trial/epidemiology of diabetes intervention and complications research group. Retinopathy and nephropathy in patients with type 1 diabetes four years after a trial of intensive therapy. N Engl J Med 2000;342:381–389.

USRDS 2009 Annual Data Report. www.usrds.org

Venkatesan B, Ghosh-Choudhury N, Das F, et al. Resveratrol inhibits PDGF receptor mitogenic signaling in mesangial cells: role of PTP1B . FASEB J. 2008 ;22(10):3469-82.

Valenzano DR. et al. Resveratrol prolongs lifespan and retards the onset of age related markers in a short-lived vertebrate. Curr. Biol. 2006;16, 296–300.

Wolf G, Sharma K, Chen Y, et al. High glucose-induced proliferation in mesangial cells is reversed by autocrine TGF-b. Kidney Int 1992; 42:647–656.

White KE, Bilous RW. Type 2 diabetic patients with nephropathy show structural-functional relationships that are similar to type 1 disease. J Am Soc Nephrol
2000;11:1667–1673.

White KE, Bilous RW, Marshall SM, et al. Podocyte number in normotensive type 1 diabetic patients with albuminuria. Diabetes 2002; 51:3083–3089.

Yue KK, Chung WS, Leung AW, etal. Redox changes precede the occurrence of oxidative stress in eyes and aorta, but not in kidneys of diabetic rats. Life Sci 2003; 73:2557– 70.

Ziyadeh FN. Mediators of hyperglycemia and the pathogenesis of matrix accumulation in diabetic renal disease. Miner Electrolyte Metab 1995; 21: 292–302.

Zou JG, Huang YZ, Chen Q,et al. Resveratrol inhibits copper ion-induced and azo compound-initiated oxidative modification of human low density lipoprotein. Biochem Mol Biol Int 1999;47:1089–96.
論文全文使用權限
  • 同意授權瀏覽/列印電子全文服務,於2013-08-06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