丙型肝炎病毒NS3基因对人肝细胞生物学特性的影响

目的:建立稳定转染HCVNS3的人源肝细胞系,探讨HCVNS3对肝细胞生物学特性的影响。方法:通过脂质体将HCVNS3的真核表达质粒稳定转染至QSG7701细胞(pRcHCNS3/QSG细胞),PCR,WesternBlot,免疫组化检测基因的整合和表达;光学显微镜和电子显微镜观察转染前后细胞超微结构的改变;流式细胞仪检测细胞周期和凋亡率变化;生长曲线,软琼脂集落实验,成瘤性实验探讨HCVNS3对肝细胞增殖的影响。结果:HCVNS3基因在pRcHCNS3/QSG细胞中得到整合和表达,定位于细胞浆。形态学显示出增殖旺盛的的特点,流式细胞仪检测pRcHCNS3/QSG细胞G0/G1期细胞数目减少而S期细胞数目增加,生长曲线和软琼脂集落实验表明pRcHCNS3/QSG细胞倍增时间明显缩短,停泊非依赖性生长能力明显增强,并能接种裸鼠成瘤,免疫组化证实肿瘤组织有HCVNS3蛋白表达。电镜和流式细胞仪检测显示HCVNS3能促进pRcHCNS3/QSG细胞凋亡。但其促增殖速度远大于凋亡率,表现为pRcHCNS3/QSG细胞获得恶性表型和致瘤性。结论:HCVNS3能明显促进人肝细胞QSG7701恶性转化,pRcHCNS3/QSG细胞可作为研究HCVNS3致癌机理的细胞模型。

Effect of Hepatitis c Virus NS3 Gene on Biological Charictarization of Human Hepatocyte

Aim:In order to construct human hepatocyte cell line which stably transfected with HCV NS3 and investigate the effect of Hepatitis c virus NS3 gene on cell biological charictarization.Methods:the HCV NS3 plasmids were stably transfected into QSG7701 cells,which was named as pRcHCNS3/QSG cells.PCR,Western Blot and immunochemistry confirmed the NS3 gene integration and expression.Light and electron microscopy observed the change of ultramicrostructure.Flow cytometry analysis detected the DNA content and apoptosis.Cell growth assay,anchorage-independent growth and tumor development experiment investigated the effect on cell proliferation.Results:HCV NS3 has been integrated and expressed in the cytoplasm of pRcHCNS3/QSG cells.The pRcHCNS3/QSG cells proliferated quickly.Flow cytometry analysis indicated that HCV NS3 could accelerate the progression of G1 to S in cell cycle.The pRcHCNS3/QSG cells showed reduced population doubling time,anchorage-independent growth and tumor development in nude mice.Immunohistochemistry confirmed the expressions of HCV NS3 proteins in tumor tissue.HCV NS3 also promoted apoptosis in pRcHCNS3/QSG cells.But the proliferation was higher than apoptosis,which resulted in malignant charaters and tumorigenic features.Conclusion:HCV NS3 can induce malignant transformation of human hepatocyte QSG7701 cells,and pRcHCNS3/QSG cells can be used as cell model for studying the mechanism of HCV NS3 carcinogenesis.