"小鼠β-1,4-N-acetyl-galactosaminyl transferase 2 於賽特利細胞之生理功能探討"

Abstract

Sda β-1,4-N-acetyl-galactosaminyl transferase Ⅱ(B4galnt2)是一醣轉移酶，主要催化轉移GalNAc以β-1,4的鍵結至[Neu5Acα2-3]Galβ1-4GlcNAcβ1-3Gal之Gal上，使醣連上形成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，為了解Sda結構的生理功能，故先了解其轉化酵素B4galnt2。由實驗室成員的研究了解B4galnt2與老鼠胚胎著床及卵母細胞成熟有關，推測Sda結構在雌性生殖系統中具有其重要性，所以了解Sda轉化酶的調控，以便推測Sda角色之可能性。實驗室已發現一賽特利細胞的細胞株(TM4)會表現B4galnt2，推測B4galnt2可能在雄性生殖系統中扮演著某種角色，至於B4galnt2的功能在雄性生殖系統尚為未知。實驗中以公鼠為動物模式利用Quantitative Real-Time PCR (qRT-PCR)及免疫螢光染色法確認初級賽特利細胞與賽特利細胞株TM4在細胞膜上有B4galnt2的存在。觀察賽特利細胞不論在繁殖作用減少時或細胞株處理follicle stimulating hormone(FSH)後增強繁殖作用後，其B4galnt2基因表現量無明顯差異，推測B4galnt2和史脫力細胞之繁殖作用較無直接相關性。利用甲狀腺激素(thyroid hormone)、視黃酸(retinoic acid)、睪固酮(testosterone)此三種賀爾蒙處理賽特利細胞株，可誘導賽特利細胞進入成熟狀態，可經成熟標記基因(maturation marker gene)及成熟後生理之特性確認其賽特利細胞之成熟。在成熟過程中，B4galnt2基因表現量上升，且利用RNA interference(RNAi)抑制基因表現實驗中發現當削弱B4galnt2表現時，成熟標記基因表現量隨之下降，顯示賽特利細胞無法進入成熟階段。綜合以上各項結果推測B4galnt2與賽特利細胞之成熟作用具有相關性，此外利用初級賽特利細胞發現小鼠在靠近青春期時，B4galnt2表現量相較於出生期高，當未成熟初級賽特利細胞誘導至成熟之初級賽特利細胞，其B4galnt2基因表現量隨之升高，故B4galnt2在小鼠體內可能也和賽特利細胞之成熟作用具有正相關性。

Sda β-1,4-N-acetyl-galactosaminyl transferaseⅡis a kind of carbohydrate transferases, whose main function is to catalyze and transfer GalNAc to Gal of [Neu5Acα2-3]Galβ1-4GlcNAcβ1-3Gal through β-1,4 bond, so that carbohydrate is linked to form the Sda structure. The structure was earliest found in the antigen structure on the blood corpuscle surface; in fact, however, it is widely distributed in the tissues and body fluids of the mammals, including stomach, intestine, kidney, serum, urine, and milk. Recent studies showed that the B4GALNT2 expression was obviously reduced in the gastric cancer and colon cancer cells of human being, causing decrease in the Sda antigen, and the said reduction was closely correlated with the metastasis of the cancer cells; this indicated the importance of B4GALNT2 and its correlation with the bio-system. However, very few studies have been conducted on its physiological functions. In 2003, the research team led by Dell first found the Sda structure in the female reproductive system. Before the physiological functions of the Sda structure was learned about, its invertase B4galnt2 needed to be studied. The research showed that B4galnt2 was correlated with the mouse’s embryo implantation and the maturation of its oocytes. It was presumed that the Sda structure played an important role in the female reproductive system. Therefore, to infer the role of the Sda structure, the regulation and control by the Sda invertase should be learned. The laboratory has found B4galnt2 in the cell strains (TM4) of the Sertoli cells, so it was supposed that B4galnt2 might play a certain role in the male reproductive system, but its functions in the male reproductive system remained unknown yet. In the experiment, a male mouse was taken as the subject. The Quantitative Real-Time PCR and immune fluorescent staining method were used to confirm the existence of B4galnt2 on the cell membranes of the primary Sertoli cells and Sertoli cell strains. The observation indicated that whether the proliferation of the Sertoli cells was declined or enhanced after the cell strains were treated with follicle stimulating hormone, the B4galnt2 gene expression showed no marked difference. In this way, it was presumed that B4galnt2 did not participate in the proliferation of the Sertoli cells. By using hormone to treat the Sertoli cell strains, the Sertoli cells might be induced into the state of maturity. The maturity of the Sertoli cells could be confirmed by the mature marker genes and post-maturation physiological features. In the process of maturation, the B4galnt2 gene expression was increased. The RNAi suppress gene expression experiment showed that when the B4galnt2 expression was reduced, the maturation marker gene expression decreased accordingly. This showed that the Sertoli cells could not enter the state of maturity. To sum up, it was presumed that B4galnt2 participated in the maturation of the Sertoli cells. In addition, the experiment on the primary Sertoli cells showed that the B4galnt2 expression in the mouse coming close to the puberty was higher than that when it was born. When the immature primary Sertoli cells were induced to be mature, the B4galnt2 gene expression in them was raised. Thus, it was presumed that B4galnt2 might participate in the maturation of the Sertoli cells in the mouse.