丙型肝炎病毒核心蛋白诱导人胆管上皮细胞转化及成瘤实验

Human normal biliary epithelial cells tranformation and tumor development induced by hepatitis C virus core protein

目的 探讨丙型肝炎病毒核心蛋白 (HCV -C)对人胆管上皮细胞 (BEC)的转化及致癌作用。方法 通过脂质体介导将含有HCV- C基因重组真核表达载体pcDNAHCV- C导入BEC细胞,G418筛选表达目的基因的细胞;聚合酶链反应 (PCR)及免疫组织化学SABC法检测细胞中HCV -C基因及蛋白的表达;细胞计数、锚着非依赖性生长实验、成瘤性检测等鉴定其生物学行为变化,免疫组织化学SABC法检测所致肿瘤组织中HCV- C蛋白表达。结果 HCV -C转染的BEC细胞中HCV- C蛋白表达于胞质,质粒pcDNAHCV C转染细胞的倍增时间较pcDNA3转染细胞和未转染BEC细胞明显缩短(分别为 14h, 28h, 30h)。pcDNAHCV C和pcDNA3转染细胞及未转染BEC在软琼脂中的克隆形成率分别为 36. 0%、2 .5%和 1 5% (P<0. 01)。3种细胞接种裸鼠后,pcDNAHCV C转染细胞注射组出现肿瘤,病理学检查证实为胆管细胞癌,肿瘤组织有HCV- C蛋白的表达。而pcDNA3转染细胞及未转染BEC细胞注射组在注射 36d后仍未见肿瘤发生。结论 HCV- C蛋白具有促胆管细胞转化和促进肿瘤发生的作用。

Objective To study the effect of hepatitis C virus core protein (HCV-C) on human normal biliary epithelial cells (BEC) transformation and tumor development. Methods BEC cells were transfected with plasmid pcDNA HCV-C (expressing HCV-C) by lipofectamine and selected in G418. The expression of HCV-C gene and protein was determined by PCR and immunohistochemical staining, respectively.Biological effect of transfected cells was observed through cell proliferation assay,anchor independent growth,and tumor development in nude mice. The expression of HCV-C protein in the induced tumor was evaluated by immunohistochemistry. Results HCV-C was strongly expressed in BEC cells transfected with plasmid pcDNA HCV-C and the positive signal was located in cytoplasm.The HCV-C expression protein in the induced cytoplasm.Cell proliferation assay showed that the population doubling time in the pcDNA HCV-C transfected cells was much shorter than that in the pcDNA3 and non-transfected cells(14 h,28 h,30 h respectively).The cloning efficiencies of transfected cells with pcDNA HCV-C, pcDNA3 and non-transfected cells were 36%,2.5% and 1.5%, respectively (P<0.01).Tumor developed in nude mice inoculated with pcDNA HCV-C transfected cells after the inoculation.HE staining showed bile duct carcinoma character and immunohistochemistry confirmed HCV-C expression in the tumor tissue.The positive control group also showed tumor development,while no tumor mass obtained in the nude mice inoculated with pcDNA3 and non-transfected cells even 36 days after the injection. Conclusion HCV-C protein showed human normal biliary epithelial cells transformation and tumorigenic features.