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目的 研究丙型肝炎病毒非结构区 3(HCVNS3)蛋白对端粒酶活性的影响 ,以探讨HCVNS3蛋白在HCV致癌中的作用 ,并观察端粒酶活性原位检测法的应用价值。方法 利用HCVNS3真核细胞表达质粒pRcHCNS3 5′(表达HCVNS3N端多肽 ) ,pRcHCNS3 3′(表达HCVNS3C端多肽 )和空白质粒pRcCMV转染NIH3T3细胞 ,分别获得 11、11和 8个阳性克隆 ;采用链霉素抗生物素 过氧化物酶法 (SP)免疫组织化学方法检测转染的NIH3T3细胞中HCVNS3蛋白表达 ,并通过端粒酶活性原位检测法和端粒酶聚合酶链反应 (PCR)酶联免疫吸附反应 (ELISA)技术分别检测转染前后NIH3T3细胞端粒酶活性的定位和定量变化。结果 HCVNS3表达质粒pRcHCNS3 5′或pRcHCNS3 3′转染的NIH3T3细胞均表达HCVNS3蛋白 ,HCVNS3蛋白阳性信号均位于细胞质中 ,并以前者表达的阳性信号为强(χ2 =6 6 6 7,P <0 0 5 ) ,各组细胞端粒酶活性存在显著差异 (F =143 0 83,P <0 0 1) ,其中质粒pRcHCNS3 5′转染的NIH3T3细胞端粒酶活性最强 ,11个克隆均呈阳性 ,质粒pRcHCNS3 3′转染的细胞次之 (P <0 0 5 ) ,空白质粒pRcCMV转染细胞和未转染NIH3T3细胞最弱 ;HCVNS3蛋白的表达水平和端粒酶活性强度之间具有显著相关性 (rs=0 80 84,P <0 0 1) ;采用端粒酶活性原位检测方法和端粒酶P
Objective To investigate the effect of HCV NS3 protein on telomerase activity in order to investigate the role of HCV NS3 protein in carcinogenesis of HCV and to observe the value of in situ detection of telomerase activity. Methods 11, 11 and 8 positive clones were obtained by transfecting NIH3T3 cells with HCV NS3 eukaryotic expression plasmid pRcHCNS3 5 ’(expressing HCV NS3 N terminal polypeptide), pRcHCNS3 3’ (expressing HCV NS3 C terminal polypeptide) and empty plasmid pRcCMV. The expression of HCV NS3 protein in transfected NIH3T3 cells was detected by immunohistochemical method and the enzyme linked immunosorbent assay (ELISA) and telomerase polymerase chain reaction (PCR) Immunoprecipitation (ELISA) techniques were used to detect the localization and quantitative changes of telomerase activity in NIH3T3 cells before and after transfection. Results HCV NS3 protein was expressed in both HCV NS3 expression plasmid pRcHCNS3 5 ’and pRcHCNS3 3’ transfected NIH3T3 cells. The positive signals of HCV NS3 protein were located in the cytoplasm, and the positive signal of the former expression was strong (χ2 = 6 6 6 7, P <0 The telomerase activity in each group was significantly different (F = 143 0 83, P <0.01). The telomerase activity of NIH3T3 cells transfected with plasmid pRcHCNS3 5 ’ (P <0.05). The expression of HCV NS3 protein and the intensity of telomerase activity were the most significant among the pRcCMV3 transfected cells and the untransfected NIH3T3 cells (Rs = 0 80 84, P <0.01). In situ detection of telomerase activity and telomerase P