CD93在細胞貼附的功能研究

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標題:	CD93在細胞貼附的功能研究 The study of biological function of CD93 on cell adhesion
作者:	李芳姿 Fang-tzu Lee
貢獻者:	陳品晟嘉南藥理科技大學：生物科技研究所
關鍵字:	細胞貼附 Adhesion CD93
日期:	2008
上傳時間:	2008-12-29 15:20:46 (UTC+8)
摘要:	CD93是一個高度醣基化的穿膜蛋白，普遍地表現在內皮細胞、嗜中性白血球、幹細胞裡。因為CD93與thrombomodulin 的結構相似；且同屬於C-type lectin-like domain superfamily。目前已知thrombomodulin 在細胞與細胞之間貼附扮演重要角色，可以透過其lectin-like domain 而影響細胞與細胞之間貼附。因此本論文主要研究與thrombomodulin結構類似的CD93在細胞貼附的生理弁遄C首先將帶有不同片段CD93基因質體，包括pEGFP-N1、pEGFP-CD93與CD93去掉細胞質尾端的pEGFP-CD93ΔC，轉染到A2058黑色素腫瘤細胞。利用共軛焦螢光顯微鏡觀察，發現CD93蛋白分佈在A2058細胞的細胞膜；尤其在細胞與細胞接觸之間更明顯，細胞型態為聚集生長。利用細胞通透性實驗方式偵測細胞間貼附緊密程度，發現過氧化酶穿透緊密單層的A2058-CD93細胞的程度比A2058-GFP和A2058-CD93ΔC低，顯示CD93蛋白表現會促進A2058黑色素腫瘤細胞的細胞間緊密接觸。利用不同的單醣或多醣測試醣類對A2058-CD93細胞聚集生長的影響，結果只有mannose可以完全破壞細胞緊密聚集，這表示mannose可能是CD93蛋白的ligand之一。接著利用calcium-switch方法觀察細胞與細胞之間貼附影響，發現加入CD93抗體或是耗盡細胞培養液中的鈣離子，都會破壞CD93蛋白媒介的A2058細胞貼附。這些現象在另一株表現內生性CD93蛋白的人類臍靜脈內皮細胞HUVEC中也可發現，顯示CD93蛋白以鈣離子依賴型式幫助維持細胞的貼附。在細胞延展實驗，發現A2058-CD93細胞的面積會比A2058-GFP大；且A2058-CD93細胞貼附也會比A2058-GFP佳。另外，為研究細胞與細胞之間的貼附過程中是否藉由actin cytoskeleton穩定弁遄A我們研究CD93與actin結合蛋白之一的ERM蛋白家族是否有交互作用。利用共同免疫沉澱分析證明CD93與ezrin會進行交互作用。利用MTT方法證明，A2058-CD93生長率會比A2058-GFP快。最後，利用Western blotting確定CD93會表現在人類臍靜脈內皮細胞HUVEC，而且，在HUVEC裡利用免疫螢光染色觀察到CD93也會與F-actin共同分佈在細胞接合處。接著用共同免疫沉澱分析證明CD93與moesin會進行交互作用。從這些結果得知，CD93蛋白可促進細胞與細胞之間的貼附，並可能透過ERM 蛋白家族連接細胞骨架來調控細胞貼附。 CD93 (C1qRp) is a transmembrane glycoprotein that is expressed on endothelial cells, neutrophils, myeloid lineage cells and haematopoietic stem cells. The structure of CD93 is similar with thrombomodulin which was reported to play an important role in mediating cell-cell adhesion through its lectin-like domain. The specific aim of this study is to investigate the role of CD93 on cell adhesion. We established green fluorescent protein-tagged CD93 or cytoplasmic domain–deleted CD93 (CD93ΔC) transfectants in A2058 melanoma. Fluorescent microscopy demonstrated that CD93-GFP distributed in the plasma membrane and particularly concentrated at cell-cell junction. A2058-CD93 cells grew as closed cluster colonies, while A2058-GFP and A2058-CD93ΔC cells grew loosely. The permeability of the monolayer of A2058-CD93 cell cultures was significantly reduced than that of the A2058-CD93ΔC and A2058-GFP cells. The cell-cell adhesion in A2058-CD93 cells was calcium-dependent and was inhibited by monoclonal antibody against CD93. These results suggested that the expression of CD93 could enhance cell-cell adhesion in a calcium-dependent manner. The effect of CD93-mediated cell adhesion was also abolished by the addition of mannose, suggesting that the carbohydrate-recognition domain of CD93 might be participated in CD93-mediated cell-cell adhesion. Determination of the spreading area of single cells indicated that the membrane extension of A2058-CD93 cells was larger than that of A2058-GFP cells. In addition, the adhesion of A2058-CD93 was more tightly than that of A2058-GFP cells. In order to elucidate whether the effect of CD93 on cell adhesion is mediated by anchoring to actin cytoskeleton, we investigated the interaction of CD93 and actin-binding protein ERM family, ezrin. We demonstrated that CD93 colocalized with ezrin at cell-cell junction on A2058-CD93 cells. We also showed that CD93 interacted with ezrin directly by co-immunoprecipitation assay. Thus, CD93 protein might connect to actin cytoskeleton by ezrin and regulated cell adhesion. Furthermore, we demonstrated that CD93- and CD93ΔC-expressed cells possessed higher proliferation rate than A2058-GFP. The CD93 distribution and its interaction with ERM protein were also confirmed on human umbilical vein endothelial cells (HUVECs). These results suggested that the expression of CD93 could enhance cell adhesion, which might be through the interaction of CD93 to F-actin by ERM protein.
關聯:	校內一年後公開，校外永不公開
顯示於類別:	[生物科技系(所)] 博碩士論文

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