中文摘要

凝血酶調節素(Thrombomodulin；簡稱TM)是一穿膜醣蛋白，其在抗凝血功能上扮演重要角色。TM已被證明除了抗凝血功能外，也有其他不同的功能。在本實驗室最近的研究發現TM經由依賴鈣離子作用lectin-like結構區，而可以媒介細胞與細胞間的聯結。先前研究提出，細胞及細胞間作用的蛋白，例如cadherin(細胞黏附蛋白)家族，在許多腫瘤細胞中，扮演生長調控者的角色。為了研究TM對細胞生長的影響，我們將A2058黑色素瘤細胞送入全長凝血酶調節素(TMG)。結果顯示送入TM全長的A2058細胞其生長速率比控制組慢。經由細胞存活度及DNA分析的結果發現，表現全長TM的A2058細胞其生長速率的緩慢，並非是經由細胞凋亡所造成。除此之外，我們利用流式細胞儀證明有表現凝血酶調節素的細胞，其細胞週期會停滯在G2/M時期。另外一方面，有表現凝血酶調節素的細胞，其細胞週期調節蛋白如：cdk1和cyclin B有隨著時間增加的趨勢；相反地，cdk2和cyclin A則沒有明顯變化；而p21及p27也有增加的趨勢。根據這些結果，我們推論凝血酶調節素藉由依賴p21CIP1/WAF1和cdk-cyclin路徑調節生長停滯。這些發現指出凝血酶調節素也許在細胞生長及細胞週期中扮演重要的角色。更進一步，這方面的研究或許能為癌症治療藥物的研發，提供一個全新的方向。

Abstract

Thrombomodulin (TM) is an integral membrane glycoprotein that has a major role in anticoagulation. It is also demonstrated that TM may possess functions distinct from its anticoagulant activity. In the previous study, we showed that TM may function as a cell-cell adhesion molecule through a Ca2+ -dependent interaction of lectin-like domain. Recent studies have suggested that cell-cell interaction proteins such as cadherin family members play important roles as growth regulators in multiple tumor types. In order to study the influence of TM on cell cycle progression, TM-negative melanoma A2058 cells were transfected with green fluorescent protein (GFP)-tagged TM (TMG). The results showed that the proliferation rate of TMG-transfected A2058 cells was slower than vector control. Cell viability assay and DNA fragmentation assay showed that different proliferation rates between A2058 control and TMG was not due to cell apoptosis. In addition, cell cycle flow cytometry analysis demonstrated that TMG-transfected A2058 cells was arrested in G2/M phase. The expression levels of G2/M phase regulated proteins, including cdk1 and cyclin B were increased in A2058 TMG cells, but cdk2 and cyclinA were not. Furthermore, the expression levels of cdk inhibitor proteins, such as p21CIP1/WAF1 and p27 were increased in A2058 TMG cells. Based on these results, we suggested that TM-mediated growth arrest was dependent on p21CIP1/WAF1 and cdk-cyclin pathway. These finding indicated that TM may play an important role in cell proliferation and cell cycle progression.

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