CD93是高度醣基化的第一型穿膜醣蛋白，它廣泛的表現在骨髓細胞、單核球細胞、內皮細胞和造血幹細胞上。在一些研究CD93功能的文獻中都推測它可能對於調節吞噬作用、細胞貼附、發炎反應有關。但至今CD93的確切功能仍然未知。CD93的結構與凝血酶調節素 (thrombomodulin；TM) 相似，同樣都具有C-type lectin like domain (CTLD)、EGF-like domain、mucin-like domain。由於TM的lectin-like domain對於調節細胞與細胞間的貼附是極為重要的區域，所以本論文的研究目的是想知道結構相似的CD93蛋白，是否具有影響細胞貼附的功能。我們利用兩種策略進行我們的研究。首先，我們透過電穿孔轉殖的方式將CD93的shRNA送入人類臍帶靜脈內皮細胞 (human umbilical vein endothelial cell, HUVEC) 中，使內生性的CD93蛋白表現量下降。然而使用CD93抗體R3及R139卻無法有效地辨識內生性CD93蛋白，於是便利用Balb/C小鼠生產能辨識CD93-D123 (CD93蛋白細胞膜外的三個區域) 的抗體，並從血清純化出抗體。結果顯示，CD93-D123抗體可以有效的辨識內生性CD93蛋白。接著，利用電穿孔轉殖的方式將shRNA送入人類臍靜脈內皮細胞，結果顯示，在mRNA和蛋白質的部份皆可看到CD93的shRNA的確可以使CD93的表現量下降。在細胞通透性的實驗，也發現CD93蛋白表現量下降後HUVEC細胞的細胞通透性較正常表現CD93蛋白的細胞來得高，顯示CD93的表現與否具有影響細胞與細胞間貼附的能力。另外，我們將CD93和去除醣類辨識區域 (CRD domain)的CD93 (CD93-△L) 基因構築在表現綠色螢光蛋白 (GFP) 的質體pEGFPN1，並轉染進人類黑色素瘤細胞株A2058中，使CD93過量表現後，再以螢光顯微鏡觀察表現CD93-GFP的細胞 (A2058-CD93)。結果發現CD93-GFP分佈在細胞膜上，且螢光大多集中在細胞和細胞接合處，生長形態為聚集生長。而表現CD93-△L的細胞 (A2058-CD93△L) 生長則較為分散。在共軛焦螢光顯微鏡的觀察中發現A2058-CD93細胞與actin骨架蛋白的位置相近，推測CD93可能經由調控actin細胞骨架而具有影響細胞貼附的功能。在細胞通透性實驗，也發現形成單層的A2058-CD93細胞的通透性較A2058-GFP、A2058-CD93△L細胞低，顯示CD93蛋白可能是透過其醣類辨識區域增強細胞與細胞間貼附的能力。在Ca2+-switch實驗，也發現到鈣離子會影響細胞的貼附，使用辨識CD93不同區域的抗體也能有效破壞細胞回復聚集的能力，推測CD93蛋白具有影響細胞貼附的功能。這些結果支持了CD93蛋白可能是透過其醣類辨識區域促進細胞與細胞間的貼附。 CD93 is a highly glycosylation type I transmembrane protein which is widely express on myeloid cells, monocytes, neutrophils, endothelial cells and haematopoietic stem cells. Researchers have speculated CD93 may involve in C1q-mediated phagocytosis, cell adhesion, and cell inflammation. The structure of CD93 is similar with thrombomodulin (TM) which was reported to play an important role in mediating cell-cell adhesion through its lectin-like domain and inhibiting cell proliferation. The specific aim of this study is to investigate the role of carbohydrate recognition domain (CRD) of CD93 on cell adhesion. Two approaches were used in this study. First, the endogenous expression of CD93 was knockdown by RNAi technology in HUVEC cells to investigate the role of CD93 on cell-cell adhesion. Second, the CD93-overexpressed A2058 cells were established for studying the effect of CD93 on cell-cell contact. The result showed that CD93 shRNA could suppress CD93 mRNA and protein level in HUVECs. The permeability of the confluent monolayer of CD93 knockdown HUVEC cells cultures was significantly higher than that of normal control HUVEC cells by transwell permeability assay. The results suggested that the knockdown of CD93 decrease cell-cell contact of HUVEC cells. In addition, we established green fluorescent protein (GFP)-tagged CD93 or CRD–deleted CD93 (CD93△L) transfectants in A2058 melanoma. Fluorescent microscopy demonstrated that CD93-GFP distributed in the plasma membrane and especially concentrated at cell-cell junction. CD93-expressed cells grew as closed cluster colonies, while CD93ΔL-expressed cells grew loosely. The permeability of the confluent monolayer of CD93 cell cultures was significantly lower than that of CD93ΔL and A2058-GFP control cells by transwell permeability assay. The results suggested that the expression of CD93 enhances A2058 cell-cell contact through its carbohydrate recognition domain. Using Ca2+-switch assay, we demonstrated that the cell-to-cell adhesion in A2058-CD93 cells was Ca2+-dependent and inhibited by anti-human CD93 antibody (R3, R139, CD93-D123, antiserum). The results showed that CD93 could function as a Ca2+-dependent cell-to-cell adhesion molecule. Taken together, the results suggested that CD93 might enhance cell-cell adhesion through its carbohydrate recognition domain.
