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

由于缺乏合适的培养体系和动物模型，阻碍了对病毒生活史和HCV感染的发病机制，特别是肝癌发生机制的深入研究。本研究利用丙型肝炎病毒（HCV）核心、E1、E2和NS 2基因，建立了Cre/loxP系统介导的HCV转基因小鼠。静脉感染表达Cre DNA重组酶的重组腺病毒（AxCANCre）可诱导HCV结构蛋白在转基因小鼠肝脏中表达。在AxCANCre输注后，对小鼠肝脏中的HCV核心蛋白产生和转基因重组进行了系列评价。转基因小鼠肝脏在3 d后，核心蛋白得到高效表达，转基因几乎完全重组，随后28 d，核心蛋白表达量和转基因重组量均持续下降。然而，30.6%的转基因重组在感染后28天仍然存在，只有2.7%的核心产量在感染后28天仍然存在。与非转基因对照组相比，转基因小鼠血清丙氨酸氨基转移酶水平在腺病毒感染后10、14和21天显著升高。组织学评分也表明腺病毒注射后转基因小鼠的肝脏发生了严重的病理变化。与非转基因对照组相比，AxCANCre输注增加了CD 8 ^+淋巴细胞向转基因小鼠肝脏的浸润。此外，细胞毒性T淋巴细胞（CTL）分离的转基因小鼠在肝损伤的HCV蛋白，E1，E2和NS 2的特异性，但不为核心蛋白。这些结果表明，HCV结构蛋白在转基因小鼠肝脏中的表达增强了肝损伤。HCV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HCV感染和HCV肝细胞癌变中的作用。

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.