臺灣博碩士論文加值系統

　　CD93蛋白是表現在單核球、嗜中性球、血小板、小神經膠質細胞、內皮細胞、早期幹細胞的第一類的細胞表面醣蛋白。人類CD93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、一個黏蛋白相似區以及一個短的細胞內尾端。CD93蛋白原本被推測與促進FcR和補體受器調控的吞噬作用相關，而命名為C1qRp。然而其他的報導認為CD93蛋白在促進吞噬作用的過程中並不需要。因此CD93蛋白的功能目前仍然是有爭議的。另一方面，CD93蛋白亦被認為參與白血球與內皮細胞的相互作用。

　　我們從人類組織細胞淋巴癌U937細胞選殖CD93蛋白的基因，並分別將CD93蛋白、刪除碳水化合物辨認區域的CD93蛋白(�尊)與刪除細胞內尾端的CD93蛋白(�媒)的基因送至A2058細胞，並挑選CD93基因穩定表現的細胞株來探討其功能。根據細胞螢光的分佈，CD93蛋白表現在細胞膜上，尤其是在微纖毛構造上，而�尊 CD93和�媒 CD93蛋白也有相似的分佈。表現CD93蛋白的A2058細胞，其延展面積會比載體控制細胞大。細胞增生速率在三種細胞皆有明顯增加，從這些結果推測CD93蛋白參與調控細胞生長及細胞延展的機制，而從�尊 CD93的結果可見，碳水化合物辨識區域也參與在這兩個機制當中，作用與CD93相似，也許與其將內部EGF相似區域更裸露出來，刺激細胞生長和延展有關；另外由�媒 CD93的結果可見，細胞內尾端對於細胞延展的促進是必要的，當尾端被刪除時，CD93對細胞延展的促進作用就和對照組無異，而在細胞生長方面，即使刪除了尾端，細胞仍然較對照組生長得快，推測是單獨的EGF相似區域就可以促進細胞生長，或是�媒 CD93的細胞外送或蛋白的結構並不能真切地模擬真實狀況。

　　此外，我們也利用人類單核球THP-1細胞株來研究CD93蛋白的功能。當THP-1細胞接受PMA刺激而分化為巨噬細胞時，CD93蛋白的表現會下降。當THP-1細胞過量表現CD93蛋白時，細胞的吞噬作用會被抑制，並且THP-1細胞吸附到內皮細胞的能力也會被抑制。這些結果顯示，CD93蛋白可能調節了單核球的某些功能。

　CD93 is a type I cell surface glycoprotein predominantly expressed on myeloid lineage cells, microglia cells, endothelial cells, and early stem cell precursors. Human CD93 consists of 631 amino acids and contains a carbohydrate recognition domain (CRD), five EGF-like domains, a mucin-like region juxtaposed to the transmembrane region, and a short cytoplasmic tail. CD93 was originally named for its suggested participation in the enhancement of FcR and complement receptor (CR1)-mediated phagocytosis. However, other reports suggested that CD93 was not required for enhancing phagocytosis in vitro. The function of CD93 remains controversial. On the other hand, recent reports have described that CD93 might mediate leukocyte – endothelial interactions.

　In this study, we cloned the gene of CD93 from human histiocytic lymphoma U937 cells and investigated the functions of CD93 in A2058 cells, which stably expressed CD93, CRD deleted CD93 (�尊 CD93) and cytoplasmic tail deleted CD93(�媒 CD93). Based on the distribution of the fluorescence, CD93 was expressed at cell membrane, especially localized at microvilli in the transfected cells. �尊 CD93 and �媒 CD93 also exhibited the similar distribution. The CD93-expressed A2058 cells spreading area were larger than vector control cells. The cell proliferation rate was significantly increased in full length CD93 expressed A2058 cells as well as �尊 CD93 and �媒 CD93 A2058 cells. These results suggested that CD93 on cell surface would regulate cell spreading and proliferation in A2058 cells.

　Moreover, the function of CD93 in THP-1 cells was also studied. We used flow cytometry to explore the expression of CD93 in PMA-stimulated THP-1 differentiation, and demonstrated that CD93 expression level was down-regulated by PMA. In THP-1 cells overexpressed CD93, the phagocytosis and adhesion to endothelial cells were suppressed. These results suggested that the expression of CD93 might regulate the functions of monocyte.

中文摘要------------------------------------- 1
英文摘要------------------------------------- 3
致謝---------------------------------------- 5
目錄---------------------------------------- 6
圖、表、附錄目錄-------------------------------- 8
縮寫檢索表----------------------------------- 10
儀器---------------------------------------- 12
藥品---------------------------------------- 14
序論---------------------------------------- 18
一、人類CD93 蛋白--------------------------- 18
二、人類CD93 蛋白的結構與功能----------------- 18
三、人類CD93蛋白與Complement component 1的關係----- 19
四、人類CD93 蛋白與人類幹細胞的關係-------------- 19
五、人類CD93 蛋白在老鼠的相似基因表現------------ 20
六、人類CD93 蛋白剔除老鼠------------------- - 20
七、人類CD93 蛋白與淋巴球的關係---------------- 20
八、人類CD93 蛋白C 端與其他蛋白的關係------------ 20
研究動機------------------------------------- 22
實驗方法------------------------------------- 23
一、人類CD93 蛋白-聚合酶連鎖反應---------------- 23
二、質體製備------------------------------- 26
三、構築質體的方法--------------------------- 29
四、細胞培養方法---------------------------- 34
五、DNA轉染系統--------------------------- 38
六、以西方墨點法確認CD93、DL CD93、DC CD93 蛋白
表現----------------------------------
40
七、細胞生長實驗(cell proliferation assay)------------- 45
八、細胞延展實驗(Cell spreading assay)------------- 46
九、流式細胞儀( Flow Cytometer)測定人類CD93 蛋白抗原
之表現--------------------------------
46
十、吞噬能力的測定( Phagocytosis assay)------------- 47
十一、經PMA 分化的轉染後THP-1 細胞黏附能力測定
( Adhesion assay of THP-1)------------------
48
十二、經PMA分化的THP-1 細胞黏附到人類臍帶靜脈內皮細
胞(HUVEC)能力測定( Assay for THP-1 adesion to
HUVEC)-------------------------------
49
實驗結果------------------------------------- 51
討論---------------------------------------- 57
參考文獻------------------------------------- 65
表----------------------------------------- 70
結果圖-------------------------------------- 71
附錄---------------------------------------- 82
自述---------------------------------------- 92

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