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研究生:湯為淳
研究生(外文):Tang Wei-Chun
論文名稱:羊膜培養環境中內皮細胞可提升整合素表現與附著度
論文名稱(外文):Endothelial Cells Cultured on Amniotic Membrane Up-regulate the Expression of Integrins and Enhance Cell Adhesion
指導教授:鄭葳鄭葳引用關係黃光裕
指導教授(外文):Vivian ChengGuang-Yuh Hwang
學位類別:碩士
校院名稱:東海大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:47
中文關鍵詞:羊膜整合素細胞貼附度
外文關鍵詞:amniotic membraneIntegrin β1cell adhesion
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  • 下載下載:14
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在心血管疾病中移植具有功能性的動脈非常迫切。在臨床上, 包括冠狀動脈與周邊血管,都需要小管徑的動脈移植。自體移植與異體移植血管的生物穩定性或是相容性在使用上須符合要求,但是這樣的來源是十分有限。而在製造人工血管的過程中,內皮細胞附著在胞外基質上是必要的先決條件。在我們的研究中,將以羊膜加工以獲得可支持內皮細胞的生長平台,並以黏著指數、反轉錄聚合連鎖反應、西方點墨法、免疫共同成沉澱與共軛焦顯微鏡去研究羊膜對於內皮細胞的影響。整合素為αβ相異二聚體之細胞底層表面受體,負責調控細胞的附著與抵抗血液的衝擊力。本實驗之目的將針對內皮細胞培養在羊膜上探討Integrin的表現與細胞黏著的影響。結果內皮細胞生長在羊膜上擁有正常內皮細胞的特色及外觀。在分子實驗的結果中,內皮細胞在羊膜上培養後,其整合素β1與活化態整合素β1的表現相對於沒生長在羊膜的內皮細胞,會有較高的蛋白質表現。細胞附著能力指數的結果,顯示黏著指數與整合素β1的表現是有正相關。
The requirements of functional artery replacement in cardiovascular diseases are urgent. Clinically, the small-diameter arteries are the standards for vascular grafts, including coronary artery bypass and peripheral vascular vessel replacement. The biostability of autografts and heterografts is satisfactory in clinical use, but the resource of the grafts is limited. Tissue-engineered blood vessels provide a promising way to generate suitable graft. The extracellular matrix is required for the adhesion of endothelial cells (ECs). Using amniotic membrane (AM), we manufactured a cell-free extracellular matrix scaffolds to obtain supportable material for endothelial cell growth. We used adhesion index, RT-PCR, western blot, immunoprecipitation, and confocal laser scanning microscopy to study the effects of AM on the endothelial cells. Integrins known as αβ-heterodimeric cell-surface basal layer receptors regulate cell adhesion and are essential for mechanotransduction of hemodynamic forces. Therefore, we investigated the effects of amniotic membrane (AM) on the expression of Integrin and EC adhesion. ECs cultured on AM exhibited normal features of the typical morphology. The molecular study of ECs cultured on AM showed a higher level of expressions in both forms of integrin β1 (integrin β1 and the active integrin β1) as compared to the ECs grown on dish without AM. We also followed the functional adhesion assay to analyze cells adhesion index. These results showed that the adhesion index and integrin β1 expression was directly related.
誌謝 ---------------------------------------------------------------------------------------- 1
目錄 ---------------------------------------------------------------------------------------- 2-3
中文摘要 ---------------------------------------------------------------------------------- 4
英文摘要 ---------------------------------------------------------------------------------- 5
實驗背景 ---------------------------------------------------------------------------------- 6-16
心血管疾病與人工血管的需求
全生物性的人工血管的架構
人工血管平台基質-羊膜
羊膜基質的內皮細胞培養環境
基底膜與細胞受器的聯結模式
胞外基質與細胞聯繫的重要性
整合素Integrins的結構與構型
羊膜與整合素Integrins與其他相似的黏著分子
活化態Integrins與細胞生存訊息傳遞
Integrins於細胞內的分子連結情形

實驗目的 --------------------------------------------------------------------------------- 17-20
探討羊膜培養環境內皮細胞穩定度
以人造沖刷力測試羊膜基質上內皮細胞的抵抗沖刷指數
以反轉錄實驗探討內皮細胞在羊膜培養環境下中整合素的連結類型
內皮細胞在羊膜培養環境下整合素與相關分子的表現量
探討內皮細胞在羊膜培養環境下細胞表面整合素表現量
探討內皮細胞中整合素在羊膜培養環境下參與細胞貼附行為
探討內皮細胞中整合素在不同培養環境下參與細胞貼附行為的比例

材料與方法 ------------------------------------------------------------------------------ 21-28
初級大動脈內皮細胞培養與動脈內皮細胞種植羊膜
核醣核酸萃取與反轉錄聚合酵素連鎖反應
以聚合酵素連鎖反應分析羊膜培養環境下Integrins表現量與時間點相關
機械力液態流動沖刷測試
細胞流式儀細胞表面蛋白質染色
分區收集沖刷測試下附著與非附著內皮細胞
蛋白質萃取與分析整合素異構體β1及其活化態的蛋白質表現
化學聯結法及免疫沉澱法
羊膜上內皮細胞的免疫螢光染色

實驗結果 --------------------------------------------------------------------------------- 29-33
羊膜培養環境下內皮細胞仍表現內皮細胞特定分子
羊膜培養環境內皮細胞附著能力指數
羊膜培養環境下內皮細胞整合素的表現
探討整合素參與細胞貼附行為
探討內皮細胞與羊膜連接處分子表現

討論 --------------------------------------------------------------------------------------- 34-40
參考文獻 --------------------------------------------------------------------------------- 41-43
圖目 --------------------------------------------------------------------------------------- 44
圖與附圖 --------------------------------------------------------------------------------- 45-55
個人資料 --------------------------------------------------------------------------------- 56-58
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