标题: | 去醣基人类滤泡刺激素之菌体表现及其对于卵巢癌细胞增生影响之研究 Bacterial Expression of Non-glycosylated Human Follicle-stimulating Hormone (NG-hFSH) and Study on the Effect of NG-hFSH on Ovarian Cancer Cell Proliferation |
作者: | 黄琮道 Tsung-Tao Huang 张正 C. Allen Chang 生物科技学系 |
关键字: | 人类滤泡刺激素;卵巢癌细胞;去醣基;菌体表现;hFSH;SKOV-3;non-glycosylated;bacterial expression |
公开日期: | 2005 |
摘要: | 人类滤泡刺激素 (hFSH) 对于卵巢癌细胞的增生存在着明确的正向促进关系,因为卵巢癌细胞上大量表现hFSH受体与其键结后导致讯息传递作用而活化癌细胞生长因子,因此hFSH与卵巢癌细胞上的受体之键结互动扮演着关键性的角色。另一方面,已知醣基支链对于滤泡刺激素讯息传递过程之重要性,若除去醣基之FSH α- 或 β-次单元体的传递活性因此而降低甚多,故醣基存在与否严重影响到FSH与其受体键结后引发的讯息传递活性。 本研究之目的为试图利用大肠杆菌重组去醣基hFSH变异体(NG-hFSH variant) ,并和野生型hFSH做卵巢癌细胞之增生活性比较,期望去醣基FSH变异体对于卵巢癌细胞的讯息传递有所影响,因而降低或压抑癌细胞的增生能力。首先利用聚合□连锁反应放大hFSHA和hFSHB两段基因,并将其分别接合到表现载体pET30a和pACYCDuet-1中,各以大肠杆菌NovaBlue (DE3) 和BL21 (DE3) 作为表现宿主,经由纯化、再折叠和双体化过程组合出预期的去醣基hFSH变异体。以MTT分析法发现到相对于野生型 (wild-type) hFSH对SKOV-3细胞株之增生活性,去醣基hFSH变异体有较低吸光值。且去醣基hFSH变异体相对于野生型hFSH有较强之受体键结力并能和野生型hFSH竞争受体产生压抑增生活性之影响。未来期望去醣基滤泡刺激素能扮演卵巢癌抑制剂或压抑卵巢癌细胞发育之重要角色。 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. |
URI: | http://140.113.39.130/cdrfb3/record/nctu/#GT009228514 http://hdl.handle.net/11536/76934 |
显示于类别: | Thesis |
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