去醣基人類濾泡刺激素之菌體表現及其對於卵巢癌細胞增生影響之研究

Abstract

人類濾泡刺激素 (hFSH) 對於卵巢癌細胞的增生存在著明確的正向促進關係，因為卵巢癌細胞上大量表現hFSH受體與其鍵結後導致訊息傳遞作用而活化癌細胞生長因子，因此hFSH與卵巢癌細胞上的受體之鍵結互動扮演著關鍵性的角色。另一方面，已知醣基支鏈對於濾泡刺激素訊息傳遞過程之重要性，若除去醣基之FSH α- 或 β-次單元體的傳遞活性因此而降低甚多，故醣基存在與否嚴重影響到FSH與其受體鍵結後引發的訊息傳遞活性。 本研究之目的為試圖利用大腸桿菌重組去醣基hFSH變異體(NG-hFSH variant) ，並和野生型hFSH做卵巢癌細胞之增生活性比較，期望去醣基FSH變異體對於卵巢癌細胞的訊息傳遞有所影響，因而降低或壓抑癌細胞的增生能力。首先利用聚合□連鎖反應放大hFSHA和hFSHB兩段基因，並將其分別接合到表現載體pET30a和pACYCDuet-1中，各以大腸桿菌NovaBlue (DE3) 和BL21 (DE3) 作為表現宿主，經由純化、再折疊和雙體化過程組合出預期的去醣基hFSH變異體。以MTT分析法發現到相對於野生型 (wild-type) hFSH對SKOV-3細胞株之增生活性，去醣基hFSH變異體有較低吸光值。且去醣基hFSH變異體相對於野生型hFSH有較強之受體鍵結力並能和野生型hFSH競爭受體產生壓抑增生活性之影響。未來期望去醣基濾泡刺激素能扮演卵巢癌抑制劑或壓抑卵巢癌細胞發育之重要角色。

Human follicle-stimulating hormone (hFSH) plays an essential role in mammalian reproduction and ovarian folliculogenesis through interaction with its specific receptor, FSHR. However, epidemiologic data have implicated higher expression of follicle-stimulating hormone receptor (FSHR) in ovarian cancerous tissues compares to normal tissues and reproductive FSH as a probable risk factor for ovarian cancer development. On the other hand, the previous studies have showed that site-directed mutagenesis identified the specific roles of the individual carbohydrate chains of FSH in signal-transducing activity and receptor-binding affinity. The binding affinity of FSH lacking any one of the oligosaccharides was increased over wild-type FSH, while the signal-transducing activity of FSH lacking the oligosaccharide at αAsn52 was markedly reduced, and that of FSH lacking either β oligosaccharide was slightly reduced. According to these previous studies, the objective of the study is to express and reconstitute Non-glycosylated hFSH in Escherichia coli and evaluate the effect of NG-hFSH on ovarian cancer cell proliferation. First, hFSHα cDNA (hFSHA) and hFSHβ cDNA (hFSHB) were amplified by PCR. Then, the products of PCR were inserted into pET30a and pACYCDuet-1, respectively. Escherichia coli NovaBlue (DE3) and BL21 (DE3) were transformed for expression hosts of hFSHA-pET30a and hFSHB-pACYCDuet-1, respectively. Through purification, refolding and dimerization, expected NG-hFSH variants were reconstituted. Using MTT and TMB assay, the proliferative and receptor-binding effects of wild-type hFSH and NG-hFSH variants on ovarian cancer cell line, SKOV-3, were identified. Finally, NG-hFSH variants have been examined that they could indeed suppress the proliferation of SKOV-3 and significantly increase the receptor binding affinity on the SKOV-3.