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系統識別號 U0026-3107201219201300
論文名稱(中文) Soluble thrombomodulin 對血管內皮細胞保護的角色
論文名稱(英文) The role of soluble thrombomodulin in endothelial protection
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) Department of Biochemistry and Molecular Biology
學年度 100
學期 2
出版年 101
研究生(中文) 林奕萍
研究生(英文) Yi-Ping Lin
學號 s16994140
學位類別 碩士
語文別 中文
論文頁數 74頁
口試委員 指導教授-李貽恆
口試委員-吳華林
口試委員-蔡曜聲
口試委員-劉秉彥
中文關鍵字 內皮細胞功能失調  細胞凋亡  可溶性凝血酶調節素  保護內皮細胞 
英文關鍵字 Endothelial dysfunction  apoptosis  soluble thrombomodulin  endothelium protection 
學科別分類
中文摘要 凝血酶調節素(Thrombomodulin,TM)為表現在內皮細胞膜上的醣化蛋白,在結構上有五個功能區,依序為lectin功能區(TMD1)、六個重複類表皮生長因子構造區(TMD2)、富含絲氨酸/蘇氨酸胺基酸區(TMD3)、穿膜蛋白區(TMD4)、及細胞質功能區(TMD5)。凝血酶調節素首先被發現主要的生理功能是和凝血酶作用成為凝血酶與凝血酶調節素複合物以活化蛋白質C,促使第五及第八凝血因子降解,以抑制凝血機制。凝血酶調節素不只表現在內皮細胞外膜,也會表現於其他細胞中;同時具有除了抗凝血機制以外的其他功能。近幾年來,我們發現於人類及老鼠的心臟肥大時,凝血酶調節素會大量表現,且可溶性凝血酶調節素可以降低心肌細胞的細胞凋亡。目前已知內皮細胞的凋亡和內皮細胞功能失調以及動脈粥狀硬化有關;而具有修復內皮細胞功能之內皮前驅細胞可以減緩動脈粥狀硬化的形成。本論文我們將探討可溶性凝血酶調節素是否影響內皮細胞凋亡,同時是否會影響內皮前驅細胞的特性。首先我們發現內皮細胞凋亡時會大量釋放凝血酶調節素至培養液中,形成可溶性凝血酶調節素;在另一盤培養的內皮細胞,外加可溶性凝血酶調節素則可有效降低細胞凋亡。可溶性凝血酶調節素促進Bcl-2家族的抗凋亡因子(Bcl-2、Bcl-xL)表現、活化AKT生存訊息傳遞路徑;並且可以有效降低促凋亡因子(Bad、Bax)表現及執行細胞凋亡因子Caspase-3、壓抑JNK死亡訊息傳遞。在另一方面,我們由健康成人血液中分離內皮前驅細胞,經可溶性凝血酶調節素處理後,可以增加內皮前驅細胞群落的數目及內皮前驅細胞的增生能力;凝血酶調節素還降低了內皮前驅細胞凋亡的情況。實驗結果顯示,除了傳統的抗凝血功能之外,可溶性凝血酶調節素可經由降低內皮細胞凋亡以及增加內皮前驅細胞的生長來保護內皮細胞,進而降低動脈粥狀硬化的風險。
英文摘要 Thrombomodulin (TM) is a cell bound transmembrane glycoprotein. Traditionally, the major physiological function of TM is to bind thrombin, and thrombin-TM complex induces the activation of protein C which inhibits coagulation by degradation of coagulation factor V and VIII. TM is expressed not only on endothelium, but also in other cell types indicating its other possible physiological functions beyond anti-coagulation. Recently, we found TM was expressed in mouse and human’s cardiomyocytes during cardiac hypertrophy and soluble TM decreases cardiomyocyte apoptosis. Endothelial cell apoptosis is associated with endothelial dysfunction which may lead to the development of atherosclerosis. Additionally, previous studies suggest that the injured endothelial monolayer is repaired by endothelial progenitor cells (EPC) which may limit atherosclerotic lesion formation. In this study, we investigate whether soluble TM has potential protective effects on endothelial cell and EPC. First, we show that stress the endothelial cell with starvation induces endothelial apoptosis and release of the soluble TM into conditioned medium. Then, the released soluble TM has protective effects on another set of cultured endothial cells to decrease endothelial cell apoptosis. Soluble TM could promote the expression level of anti-apoptotic factors in Bcl-2 family (Bcl-2, Bcl-xL) and activate the AKT survival signaling pathway. It also reduces pro-apoptosis factors (Bad, Bax), caspase-3 expression, and suppresses death signaling pathway, JNK. We also cultured EPCs which were isolated from adult blood samples. After soluble TM treatment, the colony-forming unit (CFU) and proliferation of EPC is increased. TM also decreases EPC apoptosis. In conclusion, in addition to the traditional anticoagulation function, soluble TM plays an important role in endothelium protection by reducing endothelial cell apoptosis and increasing EPC growth.
論文目次 中文摘要 2
英文摘要 3
致謝 5
目錄 6
圖目錄 8
縮寫表 9
第一章 緒論 11
第二章 研究動機與目的 20
第三章 材料與方法 21
實驗藥品 21
實驗儀器 24
實驗方法 26
1. 蛋白質來源 26
2. 細胞培養 26
A. 人類臍帶靜脈內皮細胞的採集及培養 26
B. 內皮前驅細胞的採集及培養 28
3. 蛋白質基本技術操作 29
A. 蛋白質收集 29
B. 蛋白質濃度檢測 30
C. SDS-PAGE製備 31
D. 西方墨點法 32
4. 細胞凋亡檢測 34
5. 凝血酶調節素酵素免疫分析法 35
6. 計數內皮前驅細胞群落單位 36
7. 細胞增生能力檢測 37
第四章 實驗結果 38
第五章 討論 43
第六章 參考文獻 47
結果圖表 56
附錄 70

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