丙型肝炎病毒NS3基因对人肝细胞生物学特性的影响  

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

作　　者：何琼琼[1] 肖旭贤[2] 冯德云[1] 李波[1] 郑晖[1] 程瑞雪[1] 

机构地区：[1]中南大学湘雅基础医学院病理学系,长沙410078 [2]中南大学医学化学研究中心,长沙410078

出　　处：《生物技术通报》2007年第4期135-140,共6页Biotechnology Bulletin

基　　金：国家自然科学基金"HCV蛋白介导的MAPK和NF-κB信号传导途径间cross-talk的机制研究(30270601)";"2'-5'寡核苷酸合成酶启动子介导的重组caspase-3系统治疗丙型肝炎的实验研究(30671846)资助

摘　　要：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。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.

关 键 词：丙型肝炎病毒 基因转染 生物学特性 

分 类 号：R373[医药卫生—病原生物学]