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研究生:蔡宜純
研究生(外文):Yi-Chun Tsai
論文名稱:細胞質磷脂水解酵素A2α媒介高糖導致之足細胞凋亡現象
論文名稱(外文):Cytosolic PLA2α Mediates the Apoptosis of Podocytes Induced by High Glucose
指導教授:陳鴻鈞陳鴻鈞引用關係
指導教授(外文):Hung-Chun Chen
學位類別:碩士
校院名稱:高雄醫學大學
系所名稱:醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:48
中文關鍵詞:細胞質磷脂水解酵素A2α足細胞凋亡高糖
外文關鍵詞:cPLA2αpodocyteapoptosishigh glucose
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前言:糖尿病腎病變(diabetic nephropathy)是造成已開發國家末期腎臟疾病(end-stage renal disease)的主要原因,在眾多糖尿病腎病變腎絲球的型態表現中,足細胞(podocyte)數目減少為預測糖尿病腎病變進展的重要因子,而足細胞凋亡會引起足細胞掉落。細胞質水解酵素A2 (cPLA2α) 會引起細胞凋亡、感染、發炎等現象。本實驗想要探討cPLA2α是否有參與高糖刺激足細胞凋亡的過程及其機制。
方法和材料:大鼠足細胞培養於正常糖(glucose 5.5mM)或高糖(glucose 25mM)的環境,利用免疫螢光染色、流式細胞儀、西方墨點法來觀察活性氧化物(reactive oxygen species, ROS)、凋亡現象和caspase蛋白質表現;另外在高糖組加入cPLA2α抑制劑(AACOCF3) (10μM),觀察AACOCF3在ROS、凋亡現象和caspase蛋白質表現的角色。
結果: 高糖刺激1小時後,phospho-cPLA2α/cPLA2α ratio開始增加,(control to 1 hour : 0.75±0.14 to 1.69±0.08,P<0.01),phospho-cPLA2α /cPLA2α ratio隨著時間增加 (control to 24 hour : 0.75±0.14 to 2.57±0.31,P<0.01);經過高糖刺激1小時,足細胞內ROS開始產生,出現凋亡,高糖持續刺激24小時後,大量ROS產生(control to 24 hour : 0.66±0.33 to 7.48±2.23,P<0.05),凋亡足細胞更是增加(control to 24 hour : 0.75±0.2 to 4.83±0.9,P<0.05),而caspase 3和caspase 9明顯活化(cleaved caspase 3 : control to 24 hour : 0.75±0.15 to 3.83±0.50,P<0.001;cleaved caspase 9 : control to 24 hour : 0.65±0.15 to 3.39±0.46,P<0.001),不過caspase 8對高糖的刺激沒有明顯活化反應。接著觀察cPLA2α inhibitor (AACOCF3)的影響力,在經過高糖刺激24小時後,給予AACOCF3會抑制足細胞ROS產生(HG to HG+ AACOCF3 : 3.21±0.49 to 1.34±0.08,P<0.05)和凋亡(HG to HG+ AACOCF3 : 1.76±0.20 to 1.13±0.06,P<0.05);減少caspase 3 (HG to HG+ AACOCF3 : 2.41±0.32 to 0.96±0.12,P<0.05)和9 (HG to HG + AACOCF3 : 4.76±0.35 to 2.1±0.19,P<0.01)的活化;抑制了cytochrome c的轉移到細胞質(HG to HG + AACOCF3 : 8.13±0.66 to 1.82±0.24,P<0.001),減少因高糖刺激而增加的 Bax (HG to HG+ AACOCF3 : 2.26±0.28 to 0.67±0.12,P<0.01),增加了因高糖刺激而減少的Bcl-2(HG to HG+ AACOCF3 : 0.77±0.1 to 1.34±0.06,P<0.01)。
結論: 由本實驗可證實高糖刺激足細胞凋亡的路徑為intrinsic mitochondrial proapoptotic pathway,cPLA2α經由mitochondria -dependent cascade參與高糖刺激足細胞凋亡機制。

Introduction: Podocyte depletion and apoptosis was the earliest phenomenon of progression of diabetic nephropathy. cPLA2α wound induce apoptosis in various cell model. The aim of this study was to investigate the pathway of podocyte apoptosis induced by high glucose and the role of cPLA2α in high glucose-stimulated podocyte apoptosis.
Methods & Materials: Primary rat podocytes were treated with high glucose (25mM) and AACOCF3 (10μM). Immunofluorescent studies, flow cytometry and western blotting were utilized for analysis cPLA2α phosphorylation, ROS production, apoptosis and caspase protein.
Results: After the stimulation of high glucose, cPLA2α phosphorylatio was increased at 1 hour (control to 1 hour : 0.75±0.14 to 1.69±0.08,P<0.01) and increased progressively up to 24 hours (control to 24 hour : 0.75±0.14 to 2.57±0.31,P<0.01). ROS production and apoptosis were observed at 1 hour and were augmented progressively up to 24 hours (ROS : control to 24 hour : 0.66±0.33 to 7.48±2.23,P<0.05;apoptosis : control to 24 hour : 0.75±0.2 to 4.83±0.9,P<0.05). High glucose induced the increase level of caspase 3 (control to 24 hour : 0.75±0.15 to 3.83±0.50,P<0.001) and 9 (control to 24 hour : 0.65±0.15 to 3.39±0.46,P<0.001), not caspase 8 at 24 hours. AACOCF3 attenuated ROS (HG to HG+ AACOCF3 : 3.21±0.49 to 1.34±0.08,P<0.05) production and apoptosis (HG to HG+ AACOCF3 : 1.76±0.20 to 1.13±0.06,P<0.05) induced by high glucose. The increase of caspase 3 (HG to HG+ AACOCF3 : 2.41±0.32 to 0.96±0.12,P<0.05), 9 (HG to HG + AACOCF3 : 4.76±0.35 to 2.1±0.19,P<0.01), cytosol cytochrome c (HG to AACOCF3 : 8.13±0.66 to 1.82±0.24, P<0.001) and Bax (HG to HG+ AACOCF3 : 2.26±0.28 to 0.67±0.12,P<0.01) induced by high glucose was mitigated after treatment of AACOCF3. AACOCF3 increased the diminished level of Bcl-2 elicited by high glucose (HG to HG+ AACOCF3 : 0.77±0.1 to 1.34±0.06,P<0.01).
Conclusion: High glucose-stimulated apoptosis in podocytes is mediated through intrinsic mitochondrial proapoptotic pathway. cPLA2α contributes to podocyte apoptosis induced by high glucose via mitochondria-dependent cascade.

中文摘要------------------------------------------- p.4
英文摘要------------------------------------------- p.6
前言 ------------------------------------------------ p.8
研究材料與方法---------------------------------- p.12
研究結果------------------------------------------- p.19
討論 ------------------------------------------------ p.24
結論 ------------------------------------------------ p.29
圖表 ------------------------------------------------ p.30
參考文獻------------------------------------------- p.45

1.Zimmet P, Alberti KG and Shaw J: Global and societal implications of the diabetes epidemic. Nature 414: 782-787, 2001
2.Fioretto P, Steffes MW, Brown DM and Mauer SM: An overview of renal pathology in insulin-dependent diabetes mellitus in relationship to altered glomerular hemodynamics. Am J Kidney Dis 20: 549-558, 1992
3.Pagtalunan ME, Miller PL, Jumping-Eagle S, et al.: Podocyte loss and progressive glomerular injury in type II diabetes. J Clin Invest 99: 342-348, 1997
4.Eid AA, Gorin Y, Fagg BM, et al.: Mechanisms of podocyte injury in diabetes: role of cytochrome P450 and NADPH oxidases. Diabetes 58: 1201-1211, 2009
5.Choudhury D, Tuncel M and Levi M: Diabetic nephropathy -- a multifaceted target of new therapies. Discov Med 10: 406-415, 2010
6.Mokini Z, Marcovecchio ML and Chiarelli F: Molecular pathology of oxidative stress in diabetic angiopathy: role of mitochondrial and cellular pathways. Diabetes Res Clin Pract 87: 313-321, 2010
7.Susztak K, Raff AC, Schiffer M and Bottinger EP: Glucose-induced reactive oxygen species cause apoptosis of podocytes and podocyte depletion at the onset of diabetic nephropathy. Diabetes 55: 225-233, 2006
8.Kudo I and Murakami M: Phospholipase A2 enzymes. Prostaglandins Other Lipid Mediat 68-69: 3-58, 2002
9.Chakraborti S: Phospholipase A(2) isoforms: a perspective. Cell Signal 15: 637-665, 2003
10.Pickard RT, Strifler BA, Kramer RM and Sharp JD: Molecular cloning of two new human paralogs of 85-kDa cytosolic phospholipase A2. J Biol Chem 274: 8823-8831, 1999
11.Gijon MA, Spencer DM, Kaiser AL and Leslie CC: Role of phosphorylation sites and the C2 domain in regulation of cytosolic phospholipase A2. J Cell Biol 145: 1219-1232, 1999
12.Grewal S, Herbert SP, Ponnambalam S and Walker JH: Cytosolic phospholipase A2-alpha and cyclooxygenase-2 localize to intracellular membranes of EA.hy.926 endothelial cells that are distinct from the endoplasmic reticulum and the Golgi apparatus. FEBS J 272: 1278-1290, 2005
13.Hirabayashi T and Shimizu T: Localization and regulation of cytosolic phospholipase A(2). Biochim Biophys Acta 1488: 124-138, 2000
14.Penzo D, Petronilli V, Angelin A, et al.: Arachidonic acid released by phospholipase A(2) activation triggers Ca(2+)-dependent apoptosis through the mitochondrial pathway. J Biol Chem 279: 25219-25225, 2004
15.Hayama M, Akiba S, Fukuzumi M and Sato T: High glucose-induced cytosolic phospholipase A2 activation responsible for eicosanoid production in rat mesangial cells. J Biochem 122: 1196-1201, 1997
16.Furuya Y, Tagami S, Hasegawa A, et al.: Increased glomerular cytosolic phospholipase A2 activity of OLETF rats with early diabetes. Exp Clin Endocrinol Diabetes 107: 299-305, 1999
17.Mundel P, Heid HW, Mundel TM, Kruger M, Reiser J and Kriz W: Synaptopodin: an actin-associated protein in telencephalic dendrites and renal podocytes. J Cell Biol 139: 193-204, 1997
18.Chen HC, Chen CA, Guh JY, Chang JM, Shin SJ and Lai YH: Altering expression of alpha3beta1 integrin on podocytes of human and rats with diabetes. Life Sci 67: 2345-2353, 2000
19.Lewko B and Stepinski J: Hyperglycemia and mechanical stress: targeting the renal podocyte. J Cell Physiol 221: 288-295, 2009
20.Ding G, Reddy K, Kapasi AA, et al.: Angiotensin II induces apoptosis in rat glomerular epithelial cells. Am J Physiol Renal Physiol 283: F173-180, 2002
21.Vincent AM, McLean LL, Backus C and Feldman EL: Short-term hyperglycemia produces oxidative damage and apoptosis in neurons. FASEB J 19: 638-640, 2005
22.Fiordaliso F, Leri A, Cesselli D, et al.: Hyperglycemia activates p53 and p53-regulated genes leading to myocyte cell death. Diabetes 50: 2363-2375, 2001
23.Schiffer M, Bitzer M, Roberts IS, et al.: Apoptosis in podocytes induced by TGF-beta and Smad7. J Clin Invest 108: 807-816, 2001
24.Wada T, Pippin JW, Terada Y and Shankland SJ: The cyclin-dependent kinase inhibitor p21 is required for TGF-beta1-induced podocyte apoptosis. Kidney Int 68: 1618-1629, 2005
25.Qiao X, Chen X, Wu D, et al.: Mitochondrial pathway is responsible for aging-related increase of tubular cell apoptosis in renal ischemia/reperfusion injury. J Gerontol A Biol Sci Med Sci 60: 830-839, 2005
26.Allen DA, Harwood S, Varagunam M, Raftery MJ and Yaqoob MM: High glucose-induced oxidative stress causes apoptosis in proximal tubular epithelial cells and is mediated by multiple caspases. FASEB J 17: 908-910, 2003
27.Gorin Y, Block K, Hernandez J, et al.: Nox4 NAD(P)H oxidase mediates hypertrophy and fibronectin expression in the diabetic kidney. J Biol Chem 280: 39616-39626, 2005
28.Satoh M, Fujimoto S, Haruna Y, et al.: NAD(P)H oxidase and uncoupled nitric oxide synthase are major sources of glomerular superoxide in rats with experimental diabetic nephropathy. Am J Physiol Renal Physiol 288: F1144-1152, 2005
29.Piconi L, Quagliaro L, Assaloni R, et al.: Constant and intermittent high glucose enhances endothelial cell apoptosis through mitochondrial superoxide overproduction. Diabetes Metab Res Rev 22: 198-203, 2006
30.Cai L, Li W, Wang G, Guo L, Jiang Y and Kang YJ: Hyperglycemia-induced apoptosis in mouse myocardium: mitochondrial cytochrome C-mediated caspase-3 activation pathway. Diabetes 51: 1938-1948, 2002
31.Kutuk O and Basaga H: Bcl-2 protein family: implications in vascular apoptosis and atherosclerosis. Apoptosis 11: 1661-1675, 2006
32.Uozumi N and Shimizu T: Roles for cytosolic phospholipase A2alpha as revealed by gene-targeted mice. Prostaglandins Other Lipid Mediat 68-69: 59-69, 2002
33.Breyer MD and Breyer RM: Prostaglandin E receptors and the kidney. Am J Physiol Renal Physiol 279: F12-23, 2000
34.Coffman TM, Spurney RF, Mannon RB and Levenson R: Thromboxane A2 modulates the fibrinolytic system in glomerular mesangial cells. Am J Physiol 275: F262-269, 1998
35.Pompeia C, Lima T and Curi R: Arachidonic acid cytotoxicity: can arachidonic acid be a physiological mediator of cell death? Cell Biochem Funct 21: 97-104, 2003
36.Caro AA and Cederbaum AI: Role of cytochrome P450 in phospholipase A2- and arachidonic acid-mediated cytotoxicity. Free Radic Biol Med 40: 364-375, 2006
37.Ishaque A, Dunn MJ and Sorokin A: Cyclooxygenase-2 inhibits tumor necrosis factor alpha-mediated apoptosis in renal glomerular mesangial cells. J Biol Chem 278: 10629-10640, 2003
38.Engelbrecht AM and Ellis B: Apoptosis is mediated by cytosolic phospholipase A2 during simulated ischaemia/reperfusion-induced injury in neonatal cardiac myocytes. Prostaglandins Leukot Essent Fatty Acids 77: 37-43, 2007
39.Linkous A, Geng L, Lyshchik A, Hallahan DE and Yazlovitskaya EM: Cytosolic phospholipase A2: targeting cancer through the tumor vasculature. Clin Cancer Res 15: 1635-1644, 2009

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