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系統識別號 U0026-0812200911103563
論文名稱(中文) RhoA和細胞外間質蛋白在平滑肌細胞分化指標蛋白表現上所扮演的角色
論文名稱(英文) The Roles of RhoA and Extracellular Matrix Proteins on the Expression of Smooth Muscle Differentiation Markers
校院名稱 成功大學
系所名稱(中) 生理學研究所
系所名稱(英) Department of Physiology
學年度 92
學期 2
出版年 93
研究生(中文) 洪萩菱
研究生(英文) Chiu-Ling Hung
學號 s3690102
學位類別 碩士
語文別 中文
論文頁數 72頁
口試委員 口試委員-陳麗玉
口試委員-蔡美玲
指導教授-江美治
中文關鍵字 分化指標蛋白  平滑肌細胞 
英文關鍵字 extracellular matrix  RhoA 
學科別分類
中文摘要   在血管新生和動脈粥狀硬化形成與惡化的過程中,血管平滑肌細胞會從收縮態變成合成態。目前已知不同的細胞外間質蛋白(extracellular matrix proteins)會影響平滑肌細胞的表型(phenotype),然而確實的調控機制仍不清楚;此外,研究指出小G蛋白RhoA可以藉由actin的聚合來調節平滑肌分化指標蛋白(differentiation markers)基因的表現。因此本論文的主要目的是想探討細胞外間質蛋白和RhoA在平滑肌分化指標蛋白表現上所扮演的角色。我們將繼代培養第3到第6代的大鼠主動脈血管平滑肌細胞(RASMCs)培養在有第一型膠原蛋白、Matrigel和無塗層的細胞培養皿,分別利用相位差顯微鏡觀察細胞的形態和西方點墨轉漬法偵測平滑肌分化指標蛋白的表現量。結果顯示當細胞培養在有Matrigel塗層的細胞培養皿中,細胞形態比較細長並排列成網狀,這是收縮態平滑肌細胞的一種特徵;然而以西方點墨轉漬法偵測平滑肌細胞分化指標蛋白的表現,包括 SM alpha-actin、SM-MHC及h-caldesmon,結果顯示在各組間並無顯著差異。為了進一步了解細胞外間質蛋白和平滑肌細胞表型之間的關係,我們利用RT-PCR的方法偵測Matrigel塗層組和無塗層組中細胞myocardin mRNA的表現,結果顯示在無塗層和Matrigel塗層組都有表現myocardin mRNA,顯示平滑肌分化指標蛋白表現量無差異並非細胞缺乏myocardin所致。由上述結果顯示,藉由改變細胞外間質蛋白的方式,是很難偵測出分化指標蛋白表現量的差異。另一方面,為了探討RhoA在平滑肌分化指標蛋白的表現上所扮演的角色,我們選用兩種處理方式,其一是直接將TAT-HA-RhoA V14融合蛋白(一種持續活化態的RhoA)送入Swiss 3T3和RASMCs,由流體細胞儀和免疫螢光染色的結果顯示TAT-HA-RhoA V14無法顯著增加細胞內F-actin和SM alpha-actin的表現量。其次,我們利用基因轉染使胚鼠主動脈平滑肌細胞株A10細胞表現RhoA V14,結果顯示RhoA V14會增加SM alpha-actin的表現量。總結而言,我們發現RhoA的確會影響分化指標蛋白的表現,然而Matrigel不能顯著影響平滑肌細胞的表型。
英文摘要   The phenotypic modulation of vascular smooth muscle cells (SMCs) from the contractile phenotype to the synthetic one is critically involved in vasculogenesis and in the development and progression of atherosclerosis. Extracellular matrix (ECM) proteins have been shown to promote cultured SMCs to either contractile or synthetic phenotype, but the molecular mechanisms remain largely unknown. Emerging evidence indicates that the expression of SMC differentiation markers is regulated by small G protein RhoA-mediated actin polymerization. This study was aimed to investigate the roles of ECM proteins and RhoA in the expression of SMC differentiation markers. Rat aortic smooth muscle cells (RASMCs) from passage three to six were cultured on the dishes coated with Matrigel, type I collagen or uncoated. Using phase contrast microscopy and immunoblotting analysis, RASMCs grown on Matrigel appeared thinner and longer, characteristic of contractile phenotype, than those grown on uncoated culture dishes. However, RASMCs culture with or without ECM proteins exhibited no difference in the expression of SMC differentiation markers using immunoblotting analysis. In order to clarify the mechanisms contributing to SMC phenotype regulation by ECM proteins, mRNA expression of myocardin, a critical cofactor for transcription factor serum response factor, was examined in the RASMCs cultured on dishes coated with Matrigel or uncoated. The results indicated that myocardin mRNA was expressed in RASMCs cultured under both conditions. Thus, the lack of difference in SMC differentiation markers expression does not result from the lack of myocardin expression. To examine the role of RhoA in regulating the expression of SMC differentiation markers, two sets of experiments were performed. First, a fusion protein for the constitutively active mutant of RhoA, TAT-HA-RhoA V14, was delivered to Swiss 3T3 and RASMCs. Flow cytometry and immunocytochemistry analysis showed that TAT-HA-RhoA V14 treatment at 25 nM to 200 nM did not increase F-actin and smooth muscle alpha-actin expression. Secondly, a plasmid containing GFP-RhoA V14 construct was transfected into A10 cells to examine the effects on smooth muscle alpha-actin expression. A10 cells transfected with GFP-RhoA V14 for 24 hours expressed more smooth muscle alpha-actin compared to those transfected vector alone. These results suggest that RhoA may play a role in regulating the expression of SMC differentiation markers whereas Matrigel coating does not significantly affect the phenotype of SMCs.
論文目次 誌謝.............................................II
目錄............................................III
圖表............................................V
英文摘要..........................................1
中文摘要..........................................2
緒論..............................................4
材料與方法.......................................11
一、 細胞培養...............................11
二、 細胞處理...............................13
三、 SDS-聚丙烯醯凝膠電泳分析...............14
四、 西方點墨轉漬法.........................16
五、 流體細胞偵測法.........................19
六、 免疫螢光染色法.........................21
七、 RhoA活性的分析.........................22
八、 RNA的抽取..............................24
九、 反轉錄聚合酶連鎖反應...................25
十、 數據處理與統計分析.....................26
藥品與儀器.......................................27
結果.............................................32
一、
1. 細胞外間質蛋白對RASMCs細胞形態的影響.....32
2. 細胞外間質蛋白和SFM對於分化指標蛋白表現的影
響.......................................33
3. Myocardin mRNA在RASMCs的表現.............34
二、
1. 細胞外間質蛋白對於RhoA活性的影響.........34
2. TAT-HA-RhoA V14對Swiss 3T3細胞和RASMCs F-
actin的影響..............................36
3. TAT-HA-RhoA V14對SM alpha-actin表現的影響
.........................................37
4. 以基因轉染表現RhoA V14並探討RhoA對SM alpha-
actin表現的影響..........................38
討論.............................................40
結論.............................................45
參考文獻.........................................46
圖...............................................54
作者簡歷.........................................66
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