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研究生:施孟均
研究生(外文):Meng-Chun Shih
論文名稱:探討抗氧化酵素對氧化低密度脂蛋白所引發人類主動脈平滑肌細胞增生之影響與其機制
論文名稱(外文):The effect of antioxidant enzymes on the proliferation of oxLDL-treated human aortic smooth muscle cells and its underlying mechanism
指導教授:陳玉怜
指導教授(外文):Yuh-Lien Chen
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:解剖暨細胞生物學研究所
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:2004
畢業學年度:92
語文別:中文
中文關鍵詞:平滑肌細胞氧化低密度脂蛋白超氧岐化酶過氧化氫酶細胞週期
外文關鍵詞:smooth muscle celloxLDLSODcatalasecell cycle
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血管平滑肌細胞移行至血管內層並且增生是動脈硬化和血管再狹窄的主要因素,而氧化低密度脂蛋白(oxLDL)與心血管疾病的形成息息相關;有多項證據建議,抗氧化劑可藉由減少低密度脂蛋白的氧化和抑制人類主動脈平滑肌細胞增生,來減緩心血管疾病生成。在本篇論文中欲探討過氧化氫酶(catalase)與人類銅/鋅型超氧物岐化酶(Cu/Zn SOD)的大量表現,會經由哪些機制影響oxLDL處理人類主動脈平滑肌細胞之增生。以含有人類catalase或SOD基因的腺病毒感染人類主動脈平滑肌細胞,以西方點墨法觀察到細胞質中有大量catalase 及SOD蛋白質的表現。在本研究中使用MTT assay測細胞存活率,以NADPH oxidase activity assay觀察細胞膜上NADPH oxidase的活性,利用流式細胞儀觀察細胞週期的分佈,以西方點墨法觀察細胞週期相關蛋白質、Akt訊號傳遞路徑的表現。結果發現20 □g/ml oxLDL處理人類主動脈平滑肌細胞,可提高細胞的存活率,增加細胞膜上NADPH oxidase的活性,活化Akt訊號傳遞路徑,增加cyclin D1、cyclin E、CDK2、CDK4和pRb蛋白質的表現量,刺激細胞從G1期進入S期中,促使細胞週期的進行,造成細胞增生。轉殖50 MOI的AdCAT、AdSOD或共同轉殖catalase和SOD的AdCO,可分別減少細胞受到20 □g/ml的oxLDL刺激後所引起的NADPH oxidase活性。除此之外,轉殖上述三種cDNA能抑制因oxLDL所活化的Akt訊號傳遞路徑、降低cyclin D1、cyclin E、CDK2、CDK4和pRb的表現、並藉由增加CDK抑制劑-p21、p27,減少oxLDL所造成之細胞增生現象,使細胞停滯在G0/G1期中。由上述結果推論,在動脈硬化與血管再狹窄的病理機轉中,細胞如能大量表現抗氧化酵素可減少ROS的生成,以影響細胞週期之相關蛋白質及抑制平滑肌細胞增生,達到減緩動脈硬化與血管再狹窄之進展。
The migration of smooth muscle cells (SMCs) into the intima, followed by their proliferation is a central theme of atherosclerosis and restenosis. These events are preceded and accompanied by oxidized low-density lipoprotein (oxLDL). Many lines suggest that possible effects of antioxidants on the inhibition of LDL oxidation and smooth muscle cell proliferation could play a key role in the prevention and treatment of cardiovascular disorders. The present study was designed to examine the effect and the mechanism of overexpression of antioxidant enzymes, catalase and Cu/Zn superoxide dismutase (SOD), on the proliferation of oxLDL-treated human aortic smooth muscle cells (HASMCs). HASMCs were infected with adenoviral vectors containing cDNA of human catalase (AdCAT)、 Cu/Zn SOD (AdSOD) or catalase and Cu/Zn SOD (AdCO). The infection with AdCAT or with AdSOD protein greatly increased the amount of catalase or SOD protein contents within HASMCs cytoplasm by Western blot. We examined cell survival rate by using MTT assay, activity of NADPH oxidase by NADPH oxidase activity assay, cell cycle status by flow cytometry, and expression of cell cycle related proteins、and cellular Akt by Western blot. HASMCs treated with 20 □g/ml oxLDL for 24 hours induced increase of cell viability, activation of NADPH oxidase, stimulation of G1→S cell cycle progression, activation of Akt signal pathway, and the increase of the expressing of the cell cycle inducers (cyclin D1、cyclin E、CDK2、CDK4 and pRb ). Transfection with 50 MOI AdCAT, AdSOD or AdCO in 20 □g/ml oxLDL-treated HASMCs could reduce the activation of NADPH oxidase. Besides, transfection with the three kinds of cDNA could inhibit oxLDL-activated Akt signal pathway, decrease cyclin D1、cyclin E、CDK2、CDK4 and pRb, enhace the CDK inhibitors, p21、p27, and arrest cell cycle in the G0/G1 phase in oxLDL-stimulated HASMCs. These results suggest that antioxidant enzymes may play protective roles in the pathogenesis of atherosclerosis and restenosis by reducing ROS production and inhibiting SMC proliferation.
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