Construction and analysis of viral hepatocarcinogenesis model using HCV transgenic mouse model

缺乏合适的培养系统和动物模型，阻碍了对病毒生命周期和丙型肝炎病毒感染发病机制的精细分析，特别是肝癌的发生。利用含核心、E1、E2和NS2基因的丙型肝炎病毒基因，建立了Cre/loxP系统介导的丙型肝炎病毒转基因小鼠。静脉感染表达Cre DNA重组酶的重组腺病毒(AxCANCre)诱导转基因小鼠肝脏中丙型肝炎病毒结构蛋白的表达。连续检测注射AxCANCre后小鼠肝脏中丙型肝炎病毒核心蛋白的产生和转基因重组。3d后，核心蛋白在小鼠肝脏中得到高效表达，转基因几乎完全重组，28d后核心蛋白产量和转基因重组水平持续下降。然而，30.6%的外源基因重组率在感染后28天保持，只有2.7%的核心产物在感染后28天保持。与非转基因对照相比，腺病毒感染后第10、14、21天，转基因小鼠血清丙氨酸氨基转移酶水平显著升高。组织学评分还显示，腺病毒注射后转基因小鼠的肝脏发生了严重的病理变化。与非转基因对照相比，注射AxCANCre后转基因小鼠肝脏中CD8~+淋巴细胞的渗入增加。此外，转基因小鼠肝损伤过程中分离到的细胞毒性T淋巴细胞(CTL)对丙型肝炎病毒(HCV)蛋白E1、E2和NS2具有特异性，而对核心蛋白无特异性。这些结果表明，在转基因小鼠肝脏中表达的丙型肝炎病毒结构蛋白加强了肝损伤。丙型肝炎病毒特异性CTL的功能可能是增强肝炎。该转基因小鼠模型系统使我们能够分析病毒产物和宿主免疫系统在丙型肝炎病毒感染和肝细胞癌发生中的作用。

Missing of proper culture system and animal model has hampered fine analysis of viral life cycle and pathogenesis of HCV infection, especially hepatocarcinogenesis. We established hepatitis C virus (HCV) transgenic mice mediated by Cre/loxP system using HCV cDNA including core, E1, E2 and NS2 gene. Intravenous infection of a recombinant adenovirus which expresses Cre DNA recombinase (AxCANCre) induced HCV structural protein expression in the liver of the transgenic mouse. HCV core protein production and transgene recombination in the mouse liver were serially evaluated after AxCANCre infusion. Core protein was efficiently expressed and transgene was almost totally recombinated in mouse liver after 3 days, then the levels of both core protein production and transgene recombination continuously decreased for 28 days. However, 30.6% of trasgene recombination remained at 28 days and only 2.7% of core production remained at 28 days after infection. Compared with non-transgenic controls, serum alanine aminotransferase levels of transgenic mice were significantly higher 10, 14 and 21 days after adenovirus infection. Histological scoring also indicated severe pathological changes in the liver of transgenic mice after adenovirus injection. AxCANCre infusion increased CD8^+ lymphocyte infiltration into the liver of transgenic mice compared with that of non-transgenic controls. Furthermore, cytotoxic T lymphocytes (CTLs) isolated from transgenic mice during liver injury were specific for the HCV proteins, E1, E2 and NS2 but not for core protein. These results suggest that HCV structural proteins expressed in the liver of transgenic mouse enhanced liver injury. The function of HCV-specific CTLs may be to enhance hepatitis. This transgenic mouse model system allows us to analyze the functions of viral products and host immune system in HCV infection and hepatocellular carcinogenesis of HCV.