人類凝血酶調節素調控凝血酶作用之蛋白質體分析; Proteomic Study of the Modulation of Thrombin by Thrombomodulin

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Title:	人類凝血酶調節素調控凝血酶作用之蛋白質體分析;Proteomic Study of the Modulation of Thrombin by Thrombomodulin
Authors:	王惠琳;Hui-Lin Wang
Contributors:	中國醫藥大學：醫學檢驗生物技術學系碩士班
Keywords:	凝血酶;凝血酶;調節素;Thrombin;Thrombomodulin
Date:	2008-07-25
Issue Date:	2009-08-12 17:10:01 (UTC+8)
Abstract:	凝血酶(Thrombin)是促進血管凝固的絲胺酸蛋白酶，還具有刺激細胞生長和進行生理的活性。凝血酶調控細胞生物活性，是分別透過結合凝血酶接受器(protease activated receptors,PARs)及凝血酶調節素(Thrombomodulin,TM)兩種不同的接受器，本論文主要目的在探討TM與PAR1同為thrombin的接受器，兩者之間的訊息傳導網路中的蛋白質分子上如何的變異或交互調控，以助於系統性了解thrombin的生物活性。我們利用表現PAR-1的人類胚胎腎臟細胞(Human embryonic kidney,HEK293 cells)及轉殖TM基因後的HEK293TM細胞為研究模式，觀察細胞的型態，發現HEK293TM細胞型態聚集。分析TM表現對凝血酶相關訊號機制的影響，在7.5 nM凝血酶誘導情況下，HEK293細胞在20分鐘後活化ERK，而於HEK293TM細胞中也可觀察到ERK活化且磷酸化持續至4小時。我們又利用蛋白質體技術研究表現人類TM分子前後的凝血酶對細胞表現細胞質內蛋白差異，以LC-MS/MS分析HEK293細胞質內蛋白表現圖譜，成功鑑定出558個蛋白質，並利用蛋白質分析軟體PANTHER classification system作功能性分類，共分為28類。另外利用二維電泳及DIGE分析出HEK293TM與HEK293細胞質差異蛋白表現有25個，經凝血酶調節HEK293細胞差異蛋白表現有18個，而HEK293TM差異蛋白表現有33個。以質譜儀鑑定蛋白質身分並作功能性分類，差異性蛋白多為細胞骨架蛋白，參與轉譯轉錄蛋白及參與轉譯後修飾作用。深入探討差異蛋白actin為TM response蛋白，以西方墨點法及免疫染色觀察actin多聚集於HEK293TM細胞膜上。說明TM基因改變HEK293細胞中細胞骨架蛋白的變化，我們也發現凝血酶的調控影響許多細胞骨架與代謝路徑等變化。總而言之，利用蛋白質體學觀點，可大量觀察凝血酶透過兩種接受器調控的蛋白質變化造成生物差異，也提供了深入研究的標的及廣泛的研究空間。 Thrombin, a coagulation protease, plays important roles in physiological function such as cell migration and proliferation. Protease-activated receptors 1 (PAR 1) and thrombomodulin (TM) are two different types of thrombin receptors. Comparison of thrombin-PAR1 as to their ability to induce proliferation, thrombin-TM complex seems to restrains cell proliferation. This study was designed to improve the understanding of these cross-talks between thrombin-TM and thrombin-PAR1. 7.5nM thrombin induced transient phosphorylation of ERKs in HEK293 cells with a peak at 20 min, where as overexpression of TM in HEK293 cells sustained the phosphorylation of ERKs. Increased level of ERKs phosphorylation remained on activation state at 4 hours after thrombin stimulation. We conducted a proteome analysis with cytosolic protein from PAR1-expressed human embryonic kidney 293 (HEK293) or TM- transfected HEK293 (HEK293TM) cells. We analyzed the cytosolic proteins profile of HEK293 cell through LC-MS/MS and successfully identified 558 proteins. Differentially expressed proteins in response of TM-expressed were identified by gel-based proteomics and DIGE analysis. Twenty-five protein spots associated with TM-mediated transformation were identified. These proteins were related to cell structure and motility function processing with PANTHER classification system. For thrombin-stimulated HEK293cells, proteomic analysis reproducibly revealed 51 significantly thrombin-responsive protein spots, 18 in HEK293 and 33 in HEK293TM cells. Bioinformatic annotations indicated that this set of proteins is enriched with cell structure, motility, metabolism, signal transduction, protein modification and protein post-translational processes. Protein selective identified in HEK293TM cells were analyzed by western blotting and immunohistochemistry. Actin was confirmed to be localized majored in the cell membrane of HEK293TM cells. In conclusion, we established a comparative subproteomics approach and can provide valuable information for understanding the activities of thrombin with two receptors.
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