丙型肝炎病毒非结构蛋白NS3对端粒酶活性的影响

目的研究丙型肝炎病毒非结构区3(HCV NS3)蛋白对端粒酶活性的影响,以探讨HCV NS3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HCV NS3真核细胞表达质粒pRcHCNS3-5′(表达HCV NS3 N端多肽),pRcHCNS3-3′(表达HCV NS3C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HCV NS3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 HCV NS3表达质粒pRcHCNS3-5′或pRcHCNS3-3′转染的NIH3T3细胞均表达HCV NS3蛋白,HCV NS3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=6.667,P<0.05),各组细胞端粒酶活性存在显著差异(F=143.083,P<0.01),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<0.05),空白质粒pRcCMV转染细胞和未转染NIH3T3细胞最弱;HCV NS3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=0.808 4,P<0.01);采用端粒酶活性原位检测方法和端粒酶PCR ELISA技术检测结果具有较好的一致性(rs=0.501 96,P<0.01).结论 (1) HCV NS3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;(2) HCV NS3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;(3) 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技术.

Effect of hepatitis C virus nonstructural protein NS3 on telomerase activity

OBJECTIVE: To study the effect of hepatitis C virus nonstructural protein NS3 (HCV NS3) on telomerase activity and its carcinogenesis, and to observe the practical use of in situ telomerase activity labeling. METHODS: NIH3T3 cells were transfected with plasmid pRcHCNS3-5prime prime or minute (expressing HCV NS3 C-terminal deleted protein), pRcHCNS3-3prime prime or minute (expressing HCV NS3 N-terminal deleted protein) and blank plasmid pRcCMV. Positive clones, 11 with plasmid pRcHCNS3-5prime prime or minute, 11 with pRcHCNS3-3prime prime or minute and 8 with plasmid pRcCMV were harvested respectively. Streptavidin-peroxidase conjugated method (SP) was used to detect the expression of HCV NS3 protein in NIH3T3 cells transfected with plasmid pRcHCNS3-5prime prime or minute and pRcHCNS3-3prime prime or minute. Telomerase activity was detected by in situ telomerase activity labeling method and telomerase PCR ELISA technique in the transfected and non-transfected NIH3T3 cells. RESULTS: HCV NS3 protein was expressed in the cells transfected with plasmid pRcHCNS3-5prime prime or minute or plasmid pRcHCNS3-3prime prime or minute. The positive signals of HCV NS3 protein were localized in the cytoplasm of transfected NIH3T3 cells, and the signal intensity of the former was stronger (chi(2) = 6.667 P < 0.05). There was significant difference on telomerase activity between each group (F = 134.083 P < 0.01). Telomerase activity in all 11 clones with plasmid pRcHCNS3-5prime prime or minute was stronger than cells with plasmid pRcHCNS3-3prime prime or minute (P < 0.01), whereas telomerase activity in NIH3T3 cells transfected with the plasmid pRcCMV or non-transfected NIH3T3 cells were weaker than the cells with the plasmid pRcHCNS3-3prime prime or minute. The expression level of HCV NS3 protein were correlated significantly with the strength of telomerase activity (rs = 0.8084 P < 0.01). The results obtained with in situ telomerase activity labeling corresponded to those with telomerase PCR ELISA technique (rs = 0.50196 P < 0.01). CONCLUSIONS: (1) HCV NS3 protein may activate telomerase through endogenous mechanism to induce host cells transformation. (2) The effect of HCV NS3 C-terminal deleted protein on telomerase activity in host cells may be more intense than that of HCV NS3 N-terminal deleted protein. (3) In situ telomerase activity labeling is a reliable technique for studying pathological morphology and telomerase activity in tissues and cells.