HCVNS3蛋白对正常人源肝细胞生长及MAPK磷酸化的影响

Effect of HCV NS3 on proliferation and phosphorylation of MAPK in human hepatocytes

目的:研究丙型肝炎病毒非结构区3(HCVNS3)蛋白对正常人源肝细胞的转化及MAPK磷酸化调节的作用.方法:利用脂质体介导转染技术和G418筛选得到稳定表达NS3蛋白的正常人源性肝细胞QSG7701,PCR和免疫组化S-P法检测细胞中NS3的表达;细胞记数和软琼脂实验鉴定其生物学性质;抗磷酸化MAPK抗体和抗MAPK抗体Westernblot检测转染细胞MAPK活性及表达.结果:HCVNS3转染的QSG7701肝细胞,其NS3蛋白过度表达于细胞质;真核表达质粒pRcHCNS3-5’转染细胞倍增时间为12h,较pRcHCNS3-3’、pRcCMV转染细胞和未转染的QSG7701明显缩短(分别24h,26h和28h).pRcHCNS3-5’、pRcHCNS3-3’和pRcCMV转染细胞及正常肝细胞在软琼脂中的克隆形成率分别为33%、1.33%、1.46%、1.11%,pRcHCNS3-5’形成的克隆明显高于其他三种细胞(P<0.01).pRcHCNS3-5’转染细胞MAPK磷酸化程度明显高于其他三种细胞(分别为8858±877,5612±656,2212±245和989±188).(P<0.01),而MAPK的表达量没有差异(P>0.05).结论:人源性正常肝细胞QSG7701是研究HCVNS3蛋白致病机制的较好的细胞系;HCVNS3蛋白N端多肽具有促进细胞增生和改变细胞表型的作用;HCVNS3蛋白N端多肽能上调MAPK活性,不影响MAPK的表达量.

AIM:To study effects of HCV NS3 protein on proliferation and transformation of normal human liver cell line. METHODS:QSG7701 cells were transfected with pRcHCNS3- 5’, pRcHCNS3-3’ and pRcCMV using liposome transfecting technique and selected with G418;Expression of HCV NS3 protein was determined by immunohistochemistry;Biological characters of transfected cells were evaluated by population doubling time and soft agar assays; activation of MAPK was analyzed by western blot. RESULTS:QSG7701 cells transfected with pRcHCNS3-5’ showed strong intracellular expression of HCVNS3 protein, and the positive signal was localized in cytoplasm.The level of expressed HCVNS3 protein in pRcHCNS3-3’-transfected cells was lower than that in pRcHCNS3-5’-transfected cells. The population doubling time in pRcHCNS3-5’transfected cells (12 h) was significantly shorter than that in pRcHCNS3- 3’transfected cells(24 h), pRcCMV transfected cells (26h) and normal cells (28 h) (P <0.01).The cells transfected with pRcHCNS3-5’ showed much more anchorage independent colo- nies than those with pRcHCNS3-3’ and pRcCMV (P <0.01).The cloning efficiencies of transfected cells with pRcHCNS3-5’, pRcHCNS3-3,’ pRcCMV and controls were 33 %,1.33 %, 1.46 %, 1.11 %,respectively.The level of phosphorylated MAPK incells with pRcHCNS3-5’ was much higher than those with pRcHCNS3-3’and cell transfected with pRcCMV and normal cells (8 858±877,5 612±656,2 212±245,989±188,P <0.01). CONCLUSION:QSG7701 is the good human liver cell line for investigating the pathogenesis of HCV NS3 protein.5’ region of the HCV genome segment encoding NS3 is involved in cell growth and cell phenotype. N-terminal peptide of HCV NS3 protein may up-regulate the activation of MAPK.