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研究生:陳柏谷
研究生(外文):Po-cu Chen
論文名稱:凝血酶調節素是內皮細胞上血纖維蛋白溶酶原的功能性接受器
論文名稱(外文):Thrombomodulin is a functional receptor for plasminogen on endothelial cells
指導教授:吳華林
指導教授(外文):Hua-lin Wu
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:英文
論文頁數:72
中文關鍵詞:血纖維蛋白溶酶原凝血酶調節素接受器
外文關鍵詞:Thrombomodulinplasminogenreceptor
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血纖維蛋白溶酶原(plasminogen,Plg) 是負責溶解血塊的主要蛋白分解酵素血纖維蛋白溶酶(plasmin,Plm)的前趨物,在纖維蛋白溶解作用(fibriolysis)及血塊溶解扮演重要角色。它由五個環餅結構(kringle domains)及一個催化結構域(catalytic domain)所組成。血纖維蛋白溶酶原經由血纖維蛋白溶酶原活化者(Plg activator)水解斷裂成血纖維蛋白溶酶被認為具有加速細胞移動及促進組織修復的能力,例如傷口癒合及血管新生作用。然而,在細胞周圍負責Plg/Plm與Plg結合的接受器為何及其分佈是仍然未知的。人類凝血酶調節素(thrombomodulin,TM),為穿膜醣蛋白其作為抗凝劑作用是經由凝血酶促進蛋白質C活化作用。
本篇研究,利用酵素連結免疫吸附測試法 (ELISA) 和遠西方墨點法(far Western blotting) 證明Plg直接地與TM交互作用。經由表面電漿共振試驗法 (SPR assay) 證實 Plg 與固定在晶片重組凝血酶調節素第一、二、三結構域 (TMD123) 蛋白質的分離常數是0.3 uM;去除c-Myc 及His-tag的 TMD123與固定在晶片上的Plg 蛋白質的分離常數是0.11 uM。另外,rhodamine 標記Plg (rho-Plg)可結合在人類臍靜脈內皮細胞(HUVECs)的細胞膜。經由流式細胞儀和細胞黏附實驗證實,這樣的結合方式可以被事先處理特異性的TM抗體所抑制。Plg 與TM結合同時也經由免疫共沉澱法(Co-IP)所證實。而且,rho-Plg 與TM 在HUVECs的前緣同時有局部化的現象。最後,我們亦證明TM在Plg 活化、Plg調節細胞的移動和類似毛細血管形成的演重要角色,且經由對TM的EGF相似區域的特異性抗體所證實。
綜合以上的結果,我們推斷TM在內皮細胞是Plg的接受器並且參與蛋白水解活性,且位於移動性內皮細胞的前緣,這樣的現象強調著Plg/TM在血管細胞活動及血管新生上的潛在性功能。
Plasminogen (Plg) is a precursor of plasmin (Plm), which is the major proteolytic enzyme accounting for fibrinolysis and the dissolution of blood clots. It consists of five kringle domains and a catalytic domain. The cleavage of Plg to Plm by Plg activators is believed to facilitate the cell migration and promote tissue remodeling events, such as wound healing and angiogenesis. However, the Plg receptor responsible for the pericellular distribution of Plg/Plm is still obscure. Thrombomodulin (TM), a transmembrane glycoprotein, is well characterized as an anticoagulant which functions through the thrombin-dependent protein C activation.

In this study, we showed that Plg was directly interacted with TM by ELISA and Far Western blotting. Plg bound to a recombinant TM domains 1, 2, and 3 containing c-Myc and His tags (TMD123) with a Kd of 0.30 uM; and c-Myc and His tags-deleted TMD123 (dCH-TMD123) bound to immobilized Plg with a Kd value of 0.11 uM by surface plasmon resonance (SPR) assay. In addition, rhodamine-Plg bound to the plasma membrane of human umbilical vein endothelial cells (HUVECs). The binding of Plg to HUVECs as determined by flow cytometry and cell adhesion assay was blocked by pretreatment with a TM-specific antibody. The interaction of Plg and TM was also demonstrated by co-immunoprecipitation (Co-IP) assay. Furthermore, the colocalization of rhodamine-Plg and TM at the leading edge of migrating HVUECs was also observed. Finally, the binding of Plg to cell-surface TM promoted Plg activation and Plg enhanced cell transmigration, both of which were blocked by a specific antibody against TM.Taken together, TM is a receptor for Plg in endothelial cells and participates in the localization of proteolytic activity of migrating endothelial cells at the leading edge, which highlights the potential role of Plg/TM in angiogenesis.
摘要......................................................1
Abstract .................................................2
Acknowledgements..........................................3
Index.....................................................4
Figure Contents and Appendixes............................5
Abbreviations.............................................6
Reagents..................................................7
Instruments...............................................9
Introduction.............................................10
Specific aim.............................................15
Material and Method......................................16
Result...................................................37
Discussion...............................................42
Reference ................................................46
Figure...................................................51
Appendix.................................................68
Resume...................................................72
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