丙型肝炎病毒核心蛋白基因表达致人肝细胞恶性转化的研究

丙型肝炎病毒核心（HCV-C）蛋白是维持丙型肝炎病毒结构的重要蛋白质，由于参与调节细胞的生长与凋亡，被认为与HCV感染所致的肝硬化及肝细胞癌的发生有关.为了进一步探索HCV-C蛋白与肝细胞癌发生的关系，首先构建了表达HCV-C蛋白的真核表达载体，脂质体介导转染Chang-liver人肝细胞株，建立表达HCV-C蛋白的人肝细胞模型，RT-PCR方法检测HCV-C基因在人肝细胞内的表达，蛋白质印迹和免疫细胞化学方法鉴定Chang-liver肝细胞内HCV-C蛋白及其在细胞内的分布情况.表达HCV-C蛋白的Chang-liver人肝细胞培养20代以后，与对照组细胞相比，细胞的形态出现长梭形样改变，生长速度显著加快，细胞内DNA含量的均一性变差.接种表达HCV-C蛋白的Chang-liver人肝细胞的6只裸鼠在第20天时全部有肿瘤长出，且肿瘤组织结构符合肝细胞癌病理形态特点，对照组裸鼠未见肿瘤生长.上述结果表明HCV-C基因表达可导致Chang-liver人肝细胞发生恶性转化，提示HCV-C蛋白与HCV感染所致肝细胞癌的发生有直接关系.

Research on Malignant Transformation of Cultured Human Hepatocytes by Hepatitis C Virus Core Gene Expression

Hepatitis C virus (HCV) core protein is considered an important viral structural protein and has been known to regulate proliferation and apoptosis of the cells, thus, it is belived to be related to cirrhosis of liver and hepatocarcinogenesis resulting from HCV infection. To further study the relationship between HCV core protein and hepatocarcinogenesis. The eukaryote expressing recombinant plasmid vector were constructed, then transferred it into human hepatocyte-derived Chang-liver cell line by lipofectin and established the cell model expressing HCV core protein. HCV core mRNA in the Chang-liver cells was detected by RT-PCR. Expression and distribution of the HCV core protein in the Chang-liver cells were identified by Western blot and immunocytochemistry (ICC). Phenotype of the cells expressing HCV core protein altered and displayed long fusiform shape under light microscope cultured more than 20 passages after transfection, Compared with control group cells, the cells showed a markedly increased proliferation rate, and a higher variation index of DNA content. 6/6 of BALB/c-nu/nu nude mice generated tumors until 20 days after subcutaneous inoculation of the cells expressing HCV core protein. Furthermore, constitution structure of the tumors coincided with that of hepatocarcinoma. Control group nude mice did not generated tumors. Above results indicated that HCV core protein promoted the malignant transformation of the Chang-liver cells, which implied that HCV core protein was directly related to hepatocarcinogesis result from HCV infection.