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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。方法 利用HCVNS3真核细胞表达质粒pRcHCNS3 5 ’(表达HCVNS3N端多肽 )和pRcHCNS3 3’(表达HCVNS3C端多肽 )转染NIH3T3细胞 ;采用S P免疫组化方法检测转染的NIH3T3细胞中HCVNS3蛋白表达 ,并通过端粒酶活性原位检测法、TRAP PCR和端粒酶PCRELISA技术分别检测转染前后NIH3T3细胞端粒酶活性的变化。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浆中 ,并以前者表达的阳性信号为强 ;质粒pRcHCNS3 5 ’转染的NIH3T3细胞端粒酶活性最强 (P<0 0 1) ,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 (P<0 0 5 ) ;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 (P <0 0 1) ;采用端粒酶活性原位检测方法和端粒酶PCRELISA技术检测结果具有较好的一致性。结论 HCVNS3蛋白可能是通过内源性机制激活细胞端粒酶导致宿主细胞恶性转化 ;HCVNS3蛋白N端多肽对宿主细胞端粒酶的激活作用强于C端多肽 ;进一步证实端粒酶活性原位检测法是一种适合于病理形态与功能研究的有效技术。
Objective To study the effect of HCV NS3 protein on telomerase activity in order to investigate the role of HCV NS3 protein in carcinogenesis of HCV. Methods HCV NS3 eukaryotic expression plasmid pRcHCNS3 5 ’(expressing HCV NS3 N-terminal polypeptide) and pRcHCNS3 3’ (expressing HCV NS3 C-terminal polypeptide) were transfected into NIH3T3 cells. The expression of HCV NS3 protein in transfected NIH3T3 cells was detected by SP immunohistochemistry The changes of telomerase activity in NIH3T3 cells before and after transfection were detected by in situ telomerase activity assay, TRAP PCR and telomerase PCR ELISA respectively. Results HCV NS3 protein was expressed in NIH3T3 cells transfected with HCV NS3 expression plasmid pRcHCNS3 5 ’or pRcHCNS3 3’. The positive signals of HCV NS3 protein were all located in the cytoplasm. The positive signal of the former expression was strong. NIH3T3 cells transfected with plasmid pRcHCNS3 5 ’ The telomerase activity was the strongest (P <0.01). The plasmid transfected with pRcHCNS3 3 ’was the second (P <0.05), and the plasmid transfected with pRcCMV and the untransfected NIH3T3 cells were the weakest (P <0 01) There was a significant correlation between the expression level of HCV NS3 protein and the intensity of telomerase activity (P <0.01). The results of telomerase activity in situ detection and telomerase PCRELISA assay showed good consistency. CONCLUSIONS: HCV NS3 protein may be the endogenous mechanism of activation of cell telomerase leading to malignant transformation of host cells; HCV NS3 protein N-terminal polypeptide on the host cell telomerase activation stronger than C-terminal peptide; further confirmed in situ detection of telomerase activity Act is a suitable technology for the study of pathological morphology and function.