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系統識別號 U0026-0812200913493956
論文名稱(中文) 組合蛋白αv β3 參與人類重組凝血酶調節素D23誘發的平滑肌細胞移行
論文名稱(英文) Integrin αv β3Is Involved in Recombinant Human Thrombomodulin D23-induced Smooth Muscle Cells Migration
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) of Biochemistry and Molecular Biology
學年度 95
學期 2
出版年 96
研究生(中文) 周阜儒
研究生(英文) Fu-Ju Chou
學號 s1694407
學位類別 碩士
語文別 英文
論文頁數 65頁
口試委員 指導教授-施桂月
口試委員-陳玉怜
口試委員-江美治
口試委員-吳華林
中文關鍵字 組合蛋白  平滑肌細胞 
英文關鍵字 smooth muscle cell  integrin 
學科別分類
中文摘要 凝血酶調節素(TM),表現在許多細胞表面。除了最為人所知的抗凝血活性外,凝血酶調節素可能還扮演了其他重要的生物功能。在先前的研究發現,凝血酶調節素除了表現在細胞表面,血清和尿液也有凝血酶調節素片段的存在。在這個研究中,凝血酶調節素的細胞外片段被使用來研究細胞的功能。人類重組凝血酶調節素擁有三個區域,其中第二功能區包含六個重複的類上皮生長因子(EGF-like)功能區,第三功能區則為富含絲胺酸(serine)及羥丁胺酸(Threonine)的功能區。
重組凝血酶調節素D23(rhTMD23)也可以促進血管平滑肌細胞(VSMCs)的移行。血管平滑肌是組成血管重要的成員之一,血管平滑肌的移行決定性的影響了血管內膜的增厚現象,而這個現象與血管疾病習習相關,其中包含了動脈粥狀化病變。在細胞實驗上,重組凝血酶調節素D23可以有意義的使得附著分子重新分佈至細胞前緣。而我們也證實了FAK、JNK、paxillin 等細胞附著相關的蛋白分子,會受重組凝血酶調節素D23所調控的路徑活化。除此之外,重組凝血酶調節素D23的處理也影響著基質金屬蛋白酶 (MMPs)的訊息核醣核酸表現和其酵素活性,透過這些作用會造成基質金屬蛋白酶切割細胞外基質(例如:膠原蛋白、彈性蛋白、醣蛋白) 。 在調控血管平滑肌細胞的移行的機制中,基質金屬蛋白酶表現的增加扮演著重要角色。
更進一步的我們發現,重組凝血酶調節素D23可以與組合蛋白avb3連結,並且增加組合蛋白avb3在細胞表面的表現,這個結果顯示,重組凝血酶調節素D23透過與組合蛋白avb3連結,而誘使了血管平滑肌細胞的移行。
英文摘要 Thrombomodulin (TM) is widely expressed in various cell types. Besides anti-coagulant activity, the well-known function of TM, it may have other important biological functions. The previous study showed that TM fragments are found in serum and urine. In this study, the extracellular TM domain was used to study its cellular functions. Human recombinant TMD23 domain (rhTMD23) containing six tandem epidermal growth factor-like (EGF-like) domains and Ser/Thr rich domain exhibits mitogenic activity and promote endothelial cell (EC) migration. The rhTMD23 could also promotes vascular smooth muscle cells (VSMC) migration. VSMCs are one of important members of aortic vessel and migrate to neointima crucial to the development of intimal thickening that characterizes various vascular diseases, including atherosclerosis. In vitro, rhTMD23 significantly induced redistribution of focal adhesion molecules to cell leading edge. FAK, JNK, paxillin and other focal adhesion protein are activated in rhTMD23-mediated pathways. In addition, rhTMD23 treatment could affect the mRNA expression and activity of matrix metalloproteases (MMPs); thus caused the protease cleave extracellular matrix (e.g., collagens, elastin, proteoglycans). Increased expression of MMPs plays an important role in the regulation of VSMCs migration and is involved in disease processes. Furthermore, we found that rhTMD23 could bind to avb3 integrin and increasing avb3 integrin expression on cell surface. This result suggests that avb3 integrin might midiate rhTMD23-induced VSMC migration.
論文目次 Chinese Abstract 1
English Abstract 2
Acknowledgments 3
Content 5
Abbreviation 6
Reagents 7
Figure and Table List 12
Introduction
A Thrombomodulin (TM) 13
B Structure and function of TM 14
C Atherosclerosis 15
D Vascular smooth muscle cell (VSMC) 16
E VSMC migration in Physiology  16
Specific Aim 18
Materials and Methods
Protein Purification
A Yeast Transformation 19
B Expression of recombinant TMD23  21
C Purification of TMD23 22
Molecular Technique
D Thrombin Activated Assay 24
E SDS-PAGE  25
F Western Blotting 27
G Sliver Stain 29
H Cell Culture 30
I Solid Phrase Binding Assay 33
J Imuno-fluorescence Stain 34
K Gelatin Zymography 35
L Flow Cytometric Assay 36
Result 38
Discussion 43
Reference 44
Figure and Legend 50
Table 59
Appendixes 60
Resume 65
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