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研究生:丁僑萱
研究生(外文):Chiao Hsuan Ting
論文名稱:探討Cilostazol的抗動脈粥狀硬化作用及其機轉
論文名稱(外文):Anti-atherosclerotic Effects of Cilostazol: Evidence for differentiation-promoting effects on vascular smooth muscle cells
指導教授:林光輝林光輝引用關係
指導教授(外文):K. H. Lin
學位類別:碩士
校院名稱:長庚大學
系所名稱:基礎醫學研究所
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:2008
畢業學年度:96
論文頁數:58
中文關鍵詞:動脈粥狀硬化
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Cilostazol被分為第三類phosphodiesterase的抑制劑;目前研究指出cilostazol可以藉由抑制血管平滑肌細胞 (VSMCs) 的生長進而減少動脈血管內膜的再增生。而本研究主要探討cilostazol是否可以經由改變VSMCs的表型而改善動脈血管內膜再增生的情形。
從結果發現cilostazol可以將VSMCs從serum-induced的增生表型回復為分化的表型且可促使VSMCs表現收縮蛋白。我們也發現用cAMP相似物8-bromo-cAMP處理VSMCs所得到西方點墨法的結果與cilostazol處理VSMCs時相似;而用protein kinase A (PKA) 抑制劑處理VSMCs時所得西方點墨法結果與cilostazol處理VSMCs時相反,因此我們認為cilostazol調節VSMCs收縮蛋白表現有可能是經由cAMP/PKA訊息傳遞路徑。從western blot的結果我們也發現cilostazol會促進PTEN蛋白的表現與活化cAMP response element binding protein (CREB)使其磷酸化 (p-CREB)而轉移到細胞核內。在老鼠內皮受損的模型中cilostazol可以回復原本表現低下的收縮蛋白且促使PTEN蛋白與p-CREB表現增加。
從以上結果我們認為cilostazol可以經由調節PTEN蛋白與p-CREB而使得VSMCs回復到分化的表型。而這些關於cilostazol的調節機轉對於治療動脈血管的再增生應該會有幫助。
Cilostazol, a potent type 3 phosphodiesterase inhibitor, is recently found to reduce neointimal formation and restenosis by inhibiting vascular smooth muscle cell (VSMC) proliferation. This study is to investigate whether cilostazol exerts an action on phenotypic modulation of VSMC, another important process in neointimal formation and restenosis.
VSMC were cultured in serum-containing medium. Cilostazol converted VSMCs from a serum-induced de-differentiated state to a differentiated state, as indicated by a spindle-shaped morphology and an increase in the expression of SMC differentiation marker contractile proteins. The up-regulation of contractile proteins by cilostazol involved cAMP/protein kinase A (PKA) signaling pathway, because the cAMP analog (8-bromo-cAMP) mimicked and specific PKA inhibitor opposed the effect cilostazol. Furthermore, cilostazol enhanced the expression of PTEN and activated cAMP response element binding protein (CREB) (inducing phosphorylation at Ser133 and its nuclear translocation).
In the rat carotid injury model, cilostazol reversed the down-regulation of contractile proteins and up-regulated PTEN and phosphor-CREB in the neointima induced by balloon injury. Cilostazol promotes VSMC differentiation, at least in part, through a PTEN and CREB-dependent pathway. These findings suggest a possible mechanism by which cilostazol may contribute to its beneficial effects on neointimal formation and restenosis.
指導教授推薦書………………………………………………………
口試委員會審定書……………………………………………………
誌謝…………………………..……………….………………..……….iii
中文摘要……………………………..……..…………….……………..iv
英文摘要…………………………………………………………….…..v
目錄……………………………………………………………….........vii
壹、前言…………………………………………………………………1
一、動脈粥狀硬化……………………………………………………1
二、血管平滑肌細胞…………………………………………………1
三、PDE………………………………………………………………4
四、CREB……………………………………………………….……5
五、PTEN……………………………………………………………..6
六、研究動機…………………………………………………………8
貳、材料與方法………………………………………………………....9
一、Cell culture………………….…….….…………………………..9
二、MTT assay…………………….…….……………………………9
三、Western Blot…………………….….………….………………....9
四、Promoter assay…………………….….……….………………..10
五、Immunocytochemistry……………….…….…………...…...…..11
六、In vivo……………………………….…..…..………………….12
七、數值統計………………………….…...……..……..……...…..12
參、結果………………………………………..…………..…….........13
一、Cilostazol 對血管平滑肌細胞的影響…………………….…..13
二、Cilostazol 在生物體內 (in vivo) 與體外 (in vitro)對血管平滑肌細胞表現 differentiation marker 的影響……………..….… 13
三、Cilostazol 調節血管平滑肌細胞表現 differentiation marker 的機轉..............................................................................................15
四、PTEN對於cilostazol調節血管平滑肌細胞分化的影響……17
五、CREB對於cilostazol調節血管平滑肌細胞分化的影響……18
肆、討論…………………………………………………………….....20
一、Cilostazol 對於血管平滑肌的影響………………….…......... 20
二、Cilostazol 促進血管平滑肌細胞表現收縮蛋白的機轉….......20
(一)、PTEN……………………………………………..…..…… 21
(二)、CREB……………………………………………………… 21
三、結論………………………………………………………......... 22
四、未來方向………………………………………………………. 23
伍、參考資料…………………………………………….…………… 25
陸、附錄……………………………………..……………………..…..31
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