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系統識別號 U0026-0812200915365305
論文名稱(中文) 凝血酶調節素對於血管平滑肌細胞的移行所扮演的角色
論文名稱(英文) The Role of Thrombomodulin in Vascular Smooth Muscle Cells Migration
校院名稱 成功大學
系所名稱(中) 細胞生物及解剖學研究所
系所名稱(英) Institute of Cell Biology and Anatomy
學年度 97
學期 2
出版年 98
研究生(中文) 黃彥文
研究生(英文) Yen-Wen Huang
學號 t9696401
學位類別 碩士
語文別 英文
論文頁數 55頁
口試委員 口試委員-吳華林
指導教授-江美治
口試委員-吳佳慶
口試委員-吳梨華
口試委員-李貽恆
中文關鍵字 細胞黏附  血管平滑肌細胞  凝血酶調節素  心血管疾病  細胞移行 
英文關鍵字 vascular smooth muscle cells  cell migration  thrombomodulin  cell adhesion  Cardiovascular disease 
學科別分類
中文摘要 凝血脢調節素(Thrombomodulin; TM)為一種具抗凝血功能的蛋白。近期的研究發現TM也有其他多種功能,如:促進細胞的黏附能力,抑制細胞的增生與抗發炎的功能。凝血脢調節素在正常血管的平滑肌細胞中並不表現,但在動脈粥狀硬化的血管其表現量明顯增加。目前對於凝血脢調節素在血管平滑肌細胞所扮演的功能還不清楚。因此,此研究目的為探討凝血脢調節素在血管平滑肌細胞移行所扮演的角色。本實驗使用的細胞為胚胎大白鼠主動脈的平滑肌細胞株A7r5,此細胞株不表現凝血脢調節素的訊息核醣核酸。此實驗將三種基因載體轉染入平滑肌細胞株內,使之大量表現。載體分別為連結綠色螢光蛋白的凝血脢調節素 (TMGFP)、連結綠色螢光蛋白並去除Lectin 片段的凝血脢調節素(TMGΔL)及控制組的綠色螢光蛋白(GFP)基因載體。使用傷口癒合的實驗來檢測平滑肌細胞的移行,實驗時間為12小時。實驗結果發現在低血清(0.2%)的細胞培養液或含(10%)的細胞培養液、血小板衍生性生長因子(10 ng/ml)和凝血脢(1 U/ml)刺激條件下,表現TMG的細胞的移行能力比表現GFP的細胞來得差。接下來將探討表現不同蛋白的細胞在第一型膠原蛋白和纖連蛋白(Fibronectin)的黏附能力。結果顯示,表現TM的細胞在兩種胞外間質蛋白的黏附能力較控制組高,chondroitinase(0.25 U/ml)的處理可消除兩者間的差異。此外,chondroitinase的處理亦完全抑制TM抑制細胞移行的功能,結果和黏附實驗相呼應。此研究也篩選出穩定表現TMG的細胞,並觀察TM的分布情形及細胞型態,發現TMG主要表現在細胞膜上,尤其細胞與細胞交界處最為明顯。在細胞移行能力方面,穩定表現TMG的細胞的移行能力較控制組為低,chondroitinase的處理消除兩組間的差異。為了更進一步了解TM和包外間質的關係,為了避免細胞與細胞之間的作用,將低密度篩選後的細胞種在覆蓋膠原蛋白的培養皿上並給予PDGF刺激,結果也得到表現TMG的細胞移動的能力向相較控制組來的低。接下來更進一步探討TM上的 lectin-like片段對於細胞移行和黏附能力的影響。我發現表現TMGΔL的細胞在低血清(0.2%)的細胞培養液中比表現TMG或GFP的細胞展現更高的移行能力,在其他刺激處理組,亦比表現TM的細胞更高的移行能力且和控制組無明顯差異。在chondroitinase的處理下此三組細胞之間沒有顯著差異。在細胞黏附實驗中發現表現TMGΔL的細胞比表現TM較低的黏附能力但比控制組高。我亦探討TM的表現是否影響平滑肌細胞的分化狀態。以免疫螢光染色法偵測兩種平滑肌細胞的分化指標蛋白,平滑肌專一性的肌動蛋白與SM22蛋白,結果發現表現TM的細胞其肌動蛋白纖維和SM22蛋白量較控制組少,但是整體蛋白表現量並無顯著的變化。以上的結果顯示TM抑制移行的作用主要是透過TM上的chondroitin sulfate和細胞外間質黏附接合。此外,若是去除掉TM的lectin-like片段,則使TM抑制細胞移行能力的作用消失,且促進細胞黏附胞外間質的作用也明顯下降。
英文摘要 Thrombomodulin (TM), a potent anticoagulant protein, possesses multiple functions, including cell adhesion, anti-proliferation and anti-inflammation. TM is not expressed in vascular smooth muscle cells (VSMC) of healthy arteries but is upregulated in VSMC of atherosclerotic vessels. Whether TM expressed in VSMC plays functional roles during atherosclerotic progression remains unclear. This study was investigated the role of TM in VSMC migration. Gene constructs containing TM and green fluorescence protein (TM-GFP, abbreviated as TMG), lectin-like domain-deleted TM and GFP (TMGΔL) and GFP alone (as the control) were transfected into A7r5 cells, a rat aortic smooth muscle cell line that does not express TM mRNA. The VSMC migration was examined with wound healing assay for a 12-hour period. The results showed the TM-expressing VSMC exhibited lower migration activity than GFP-expressing VSMC under serum starvation (0.2%) or stimulation with serum (10%), PDGF (10 ng/ml) and thrombin (1 U/ml). Using adhesion assay, TM expression markedly enhanced cell adhesion to type I collagen or fibronectin and chondroitinase (0.25 U/ml) treatment eliminated this effect. In accordance with these results, chondroitinase eliminated TM inhibitory effect on VSMC migration. In A7r5 clones stably expressing TM, we examined TM distribution and cell morphology. TM was mainly localized on cell surface and appeared most prominent at cell-to-cell contact sites. In wound healing assay, the TMG-expressing cells had lower mobility than the control cells under serum starvation, the effect being eliminated again by chondroitinase treatment. To eliminate the effect of cell-to-cell contact on migration, we performed migration assay using cells plated at low density (2 x 103/cm2). Similar results were obtained under this condition, suggesting that TM inhibited cell migration through mechanisms other than increasing the cell-to-cell contact. Next, we examined the role of lectin-like domain of TM in cell mobility and adhesion. The results showed that the TMGΔL-expressing cells exhibited markedly higher migration activity than both the TMG-expressing cells and the control cells under serum starvation. Under stimulation, the TMGΔL-expressing cells had higher mobility than TM-expressing cells but did not differ from the control cells. There was no difference in cell mobility between GFP-, TMG- and TMGΔL-expressing cells after chondroitinase treatment. Furthermore, we examined whether TM regulated VSMC differentiation. Examined with immunofluorescence, two differentiation makers of VSMC, SM22 and smooth muscle-specific α-actin, were decreased in the TMG-expressing cells without changes in overall protein levels. These results indicated that TM attenuated VSMC migration, at lease in part, through chondroitin sulfate-mediated cell adhesion to extracellular matrices. In addition, the deletion of lectin-like domain eliminated TM inhibition on mobility and decreased TM enhancement on cell adhesion to ECM.
論文目次 Table of Contents
Chinese Abstract………………………………………… II
Abstract………………………………………………… IV
Introduction……………………………………………… 1
Materials and Methods………………………………… 9
Results…………………………………………………… 22
Discussion………………………………………………… 28
References………………………………………………… 32
Figures………………………………………………………43
Appendix 1………………………………………………… 54
Appendix 2………………………………………………… 55
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