| 摘要: | 人類CD93蛋白是一種位於細胞表面的穿膜醣蛋白，具有652個胺基酸，其中包含了碳水化合物辨認區域、五個EGF 相似區域、一個黏蛋白相似區域以及一個短的細胞內尾端。CD93蛋白最初被認為和補體分子C1q促進的吞噬作用有關，然而最近的一些研究卻認為CD93蛋白並不參與C1q促進的吞噬作用。因此CD93蛋白的功能目前仍然是有爭議而且是未知的。本計畫將以人類黑色素瘤細胞A2058 與人類臍靜脈內皮細胞 HUVECs為研究對象，探討CD93在細胞型態、黏附、增生與移行的功能。第一年擬將建構好的全長CD93 基因，碳水化合物辨認區域缺失(CD93DL)、EGF 相似區域缺失 (CD93DD2)、黏蛋白相似區域缺失(CD93DD3)、細胞內尾端缺失(CD93DC)、以及細胞內尾端突變的CD93 之cDNA構築在綠色螢光蛋白GFP表現載體pEGFPN1，轉染至A2058 細胞並挑選出穩定表現株。再利用這些細胞株研究細胞型態、黏附、增生與移行的變化，以推論CD93 蛋白與其區域片段的功能。第二年將CD93 的shRNA 轉染至HUVECs來抑制CD93的表現，並觀察細胞型態、黏附、增生與移行的變化，以探討CD93在血管內皮細胞的功能。CD93 蛋白調控細胞型態變化的機制亦進行探討，包括分析CD93 蛋白與ERM蛋白的交互作用，以及對於small GTPase RhoA家族的活化等。並研究CD93 蛋白可能透過哪些訊號傳遞機制影響細胞的增生與移行。第三年將製備CD93細胞外區域的重組蛋白，以進行CD93 蛋白間的結合能力測試。也擬利用CD93 的shRNA 降低單核球細胞的CD93蛋白表現，來探討CD93 蛋白表現對吞噬作用的影響。最後，擬將表現CD93 的A2058 細胞注射入老鼠體內，研究CD93 在活體動物內對腫瘤形成的影響。此研究計畫的執行成果將使我們對CD93 的生理功能有更深入的瞭解，同時可探討 CD93 蛋白之不同區域所扮演的角色及可能的生理意義，並且從中開發出CD93 蛋白在生物醫學上的應用價值。
Human CD93 is a cell surface transmembrane glycoprotein, which consists of 652 amino acids and contains a carbohydrate recognition domain (CRD), five epidermal growth factor (EGF)-like domains, a mucin-like region juxtaposed to the transmembrane region, and a short cytoplasmic tail. CD93 was originally suggested to participate in the enhancement of complememt receptor 1 (CR1)-mediated phagocytosis. However, other reports suggested that CD93 was not required for enhancing phagocytosis. Therefore, the functions of CD93 remain controversial and unknown. This project will use human melanoma A2058 cells and human umbilical vein endothelial cells (HUVECs) as models to investigate the roles of CD93 on cell morphology, adhesion, proliferation and migration. In first year, the green fluorescent protein (GFP) expression vector pEGFPN1 will be constructed with full-length CD93, CRD-deleted- (CD93DL), EGF-like domain-deleted (CD93DD2), mucin-like domain-deleted- (CD93DD3) and cytoplasmic tail-deleted (CD93DC), cytoplasmic domain mutated CD93. These CD93 expression vectors will be then transfected to A2058 cells for selecting stable clones. The cell morphology, adhesion, proliferation and migration of these CD93 stably expressed A2058 cells will be determined and the functions of CD93 and its domains will be investigated. In second year, we will utilize CD93 shRNA to knock-down the CD93 expression in HUVECs, and examine the cell morphology, adhesion, proliferation and migration for elucidating the role of CD93 on HUVECs. The mechanisms of CD93 regulates cell morphology will also be investigated, including CD93-ERM interaction and small GTPase RhoA family activation. In addition, we will investigate the signaling pathways that regulate cell proliferation and migration in CD93-expressed A2058 cells and CD93-knock-down HUVECs.In third year, we will prepare recombinant CD93 domains for testing the homotypic binding activity of CD93 protein. Moreover, we will use CD93 shRNA to knock-down the CD93 expression in THP-1 cells, and determine the role of CD93 expression on phagocytosis. Finally, CD93-expressed A2058 cells will be injected to SCID mice for studying the effects of CD93 on tumor growth in animal model. From the results of this project, we will further understand the biological functions of CD93. Through analyzing the effects of CD93 and CD93 domains, we can evaluate the roles and the physiological functions of different CD93 domains. These results may create the biomedical applications of CD93 protein. |
