- 學位論文
小鼠β-1,4-N-acetyl-galactosaminyl transferase Ⅱ 蛋白在卵巢的功能探討
Functional characterization of β-1,4-N-acetyl-galactosaminyl transferase Ⅱ protein in mouse ovary
摘要
Sda β-1,4-N-acetyl-galactosaminyl transferase Ⅱ(β4galNAcTⅡ,人類中為B4GALNT2；老鼠為B4galnt2)此一醣轉移酶主要催化轉移GalNAc以β-1,4的鍵結，至[Neu5Acα2-3]Galβ1-4GlcNAcβ1-3Gal之Gal上，形成Sda結構。此一結構最早是在血球表面抗原結構所發現，實際上它廣泛存在於哺乳動物組織或體液中，包含胃、腸、腎、血清、尿液和乳汁等，近期研究發現人類胃癌和結腸癌細胞中B4GALNT2表現量會明顯下降，而使Sda抗原減少並與癌細胞轉移有密切關係。由此可知B4GALNT2的重要性與生物系統的相關性，但目前為止對其生理功能研究仍有很大的空間。2003年Dell的研究團隊首先在雌性生殖系統中發現Sda結構的存在，並由實驗室成員的研究了解B4galnt2和老鼠胚胎著床有關。本論文希望能進一步探討B4galnt2在其它雌性生殖器官中的生理特性，以便深入了解Sda結構在生殖系統中的重要性。 實驗以雌鼠為模式動物，首先確定B4galnt2在卵巢的表現，並測得Sda結構在卵子中存在，卻未能在卵丘細胞上表現Sda結構，進一步觀察B4galnt2在卵巢所排出的卵丘卵母細胞複合體(COCs)的表現位置，發現B4galnt2表現在卵丘細胞的細胞膜上。利用B4galnt2抗體觀察到B4galnt2可能對卵丘卵母細胞複合體中的卵子發育(oogenesis)成熟的關鍵步驟─卵丘擴張作用(cumulus expansion)有促進作用，綜合證據顯示卵丘擴張作用不僅需要促濾泡成熟激素(FSH)也需要B4galnt2蛋白存在。推測B4galnt2於卵丘擴張作用中與細胞移動有關，進一步探討B4galnt2和細胞移動能力的關連，利用以siRNA重組質體轉染有表現B4GALNT2之結腸癌細胞株HCT116測得細胞移動的降低，但抗體對其移動能力卻無影響；同時以HCT116、CaCo-2、SW480細胞株探討B4GALNT2抗體對於細胞移動能力的影響，結果也顯示無明顯差異。
並列摘要
Sda β-1,4-N-acetyl-galactosaminyl transferase Ⅱ(β4galNAcTⅡ,B4GALNT2 in human and B4galnt2 in mouse) catalyze the addition of GalNAc to the Gal of the [Neu5Acα2-3]Galβ1-4GlcNAcβ1 -3Gal terminal structure via the β-1,4 linkage to form Sda antigen. The Sda antigen was first reported as a human blood surface antigen, and in the later researches, it was found in several tissue types, including stomach, colon, kidney, and oocytes. Its presence was also demonstrated in various body fluids, such as saliva, milk, serum and urine. Current studies revealed that Sda antigen reduced remarkably in cancer lesions of the gastrointestinal tract. It indicated the significance of this antigen in biosystem and also associated it is importance to B4galnt2. In 2003, Dell’s group found Sda antigen in reproductive system firstly, however, the significance of Sda is still unclear. My research intends to approach biological characteristics of B4galnt2, and on it, can investigate physiological role of Sda in reproductive system. Female ICR mouse is selected as an animal model. B4galnt2 expression was detected in ovary, and Sda antigen was further confirmed in oocyte but not in cumulus cell. Based on that, Immunohistochemical analysis was used to investigate B4galnt2 distribution in whole cumulus-oocyte complexes (COCs) , and the B4galnt2 existing on plasma membrane of cumulus cell was observed. Blocking the COC cultivation with B4galnt2 antibody resulted in disturbing of cumulus expansion, and it indicated that B4galnt2 protein is essential for cumulus expansion, which is a critical step during oogenesis. Cumulus expansion depends on both FSH in the environment and B4galnt2 protein that exist on the cell membrane of cumulus cell, and may be associated with cell migration ability. B4GALNT2 silencing of HCT116 with transfecting the plasmid contained B4GALNT2-siRNA caused the cell migration slower, but blocking with antibody made no difference, and the similar result was obtained from the two other colonic cell-CaCo2 and SW480 in antibody blocking test.
參考文獻
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