楊西苑臺灣大學:分子與細胞生物學研究所周耀文Chou, Yao-WenYao-WenChou2010-06-022018-07-062010-06-022018-07-062009U0001-1701200902222900http://ntur.lib.ntu.edu.tw//handle/246246/184713動脈硬化發生時，於動脈新內層 (neo-intima)有新內層平滑肌細胞增生，而新內層的平滑肌細胞不同正常成熟血管平滑肌細胞，會失去收縮能力，並有了增生、移動能力和分泌大量胞外基質，此血管新內層之平滑肌型態的特異是一個重要的課題。巢蛋白是一類中間絲蛋白，專一表現在肌肉和神經的幹細胞。而本研究利用大鼠主動脈血管平滑肌細胞當做實驗材料，並以免疫螢光染色方法發現有分泌型特徵的血管平滑肌細胞會表現巢蛋白；而將細胞培養於去除血清之培養液中，血管平滑肌細胞會分化為具有收縮型平滑肌細胞的特徵，然而巢蛋白的表現明顯減弱。這個結果也暗示了巢蛋白是分泌型血管平滑肌細胞的標誌蛋白。為了要找出調節巢蛋白表現的胞外因子，利用免疫螢光染色對細胞培養的血管平滑肌細胞來做研究，結果顯示，在細胞培養中，表皮生長因子可誘導巢蛋白表現。進一步做訊息傳遞分析，顯示出表皮生長因子主要藉由活化Ras-Raf-ERK訊息路徑來誘導巢蛋白的再表現。而這是第一個研究指出在血管平滑肌細胞中，巢蛋白可藉由胞外的生長因子刺激調節。During the progression of atherosclerosis, vascular smooth muscle cells (VSMCs) are observed in the arterial neo-intima where they proliferate. The neo-intimal smooth muscle cells are different from the mature VSMCs in that they lack of the contraction abilities, but display the characteristics of proliferation, migration, as well as the ability to secrete a large amount of extra-cellular matrix. The formation of this special phenotype of VSMCs in the neo-intima layer is a key event during the atherosclerosis.estin is a intermediate filament (IF) protein specifically expressed in myogenic and neural stem cells. In this study, we showed that nestin is expressed in primary cultures of rat aortic VSMCs that represent the synthetic phenotype and its expression diminished in those cells upon differentiation to the contractile phenotype after serum deprivation.These results suggested that nestin is a marker of synthetic VSMCs. To identify the extracellular factors that regulate nestin expression, immunofluorescence microscopy was used. Results showed that epidermal growth factor (EGF) induced nestin expression in serum-deprived primary cultures of VSMCs. Furthermore, signaling analyses revealed that the EGF-induced nestin re-expression is mediated through the activation of the Ras-Raf-ERK signaling axis. This is the first report showing that nestin expression is regulated by the extracellular growth factors in VSMCs.頁數謝…………………………………………………………………………......i文摘要……………………………………………………………………….ii文摘要………………………………………………………………………iii次……………………………………………………………………………iv論言......................................................................................................................1管平滑肌細胞的型態轉變和動脈硬化關係..................................................2管平滑肌型態的標誌蛋白..............................................................................6胞骨骼..............................................................................................................9間絲蛋白.........................................................................................................11estin...................................................................................................................14GF和EGF receptor...........................................................................................18GF receptor之傳導路徑...................................................................................20驗目的.............................................................................................................23驗材料 驗細胞.............................................................................................................24胞培養材料.....................................................................................................24疫螢光染色材料.............................................................................................25體.....................................................................................................................25品.....................................................................................................................26驗方法大鼠主動脈血管平滑肌細胞進行細胞培養..................................................27管平滑肌細胞培養..........................................................................................28疫螢光顯微鏡技術學......................................................................................28胞藥劑處理......................................................................................................29測細胞增生處理..............................................................................................29胞免疫螢光染色資料統計分析......................................................................30驗結果鼠主動脈血管平滑肌細胞的確認與觀察.....................................................30測血管平滑肌細胞之nestin表現..................................................................31管平滑肌細胞進行serum free處理之型態改變..........................................31estin在血管平滑肌細胞進行serum free處理之表現變化血管平滑肌細胞型態轉變之關係................................................................32GF誘導serum free後的血管平滑肌細胞之nestin再表現.........................33GF 促進nestin再表現之濃度效應...............................................................34GF對血管平滑肌細胞之nestin表現和其型態轉變影響............................34GF 藉由活化EGF receptor來誘導nestin再表現.......................................36GF誘導nestin的表現需要經由蛋白質新生................................................37GF誘導nestin的再表現主要藉由Ras-Raf 路徑........................................37GF促進nestin的再表現主要藉由Ras-Raf-ERK 路徑..............................38論管平滑肌細胞nestin的調控.........................................................................40管平滑肌細胞型態轉變與其nestin表現之關聯.........................................43estin對血管平滑肌細胞之可能功能探討.....................................................44estin之表現和動脈硬化的關聯探討.............................................................45來展望............................................................................................................48考文獻..................................................................................................................49圖...........................................................................................................................61136 bytestext/htmlen-US血管平滑肌細胞型態巢蛋白表皮生長因子VSMCsphenotypenestinEGFhttp://ntur.lib.ntu.edu.tw/bitstream/246246/184713/1/index.html