Analysis of viral counter regulation against host's defense mechanism for viral infection 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。我们利用Cre/loxP开关系统建立了丙型肝炎病毒转基因小鼠模型。我们将新霉素抗性基因作为填充物，在CAG启动子和核心之间的5‘和3’端分别加上loxP序列，插入到NS2、HCVcDNACN2中。取CN2转基因小鼠的脾组织。用表达cre DNA重组酶的重组腺病毒(AxCANCre)感染小鼠脾细胞和成纤维细胞，免疫印迹和免疫染色结果显示，在8个细胞系中，有2个细胞系的脾细胞和成纤维细胞高表达CORE、E1和E2蛋白。为了诱导丙型肝炎病毒在体内和肝脏中的表达，我们从尾静脉注射AxCANCre给CN2转基因小鼠，证实了其肝脏组织中有丙型肝炎病毒蛋白的表达。随着丙型肝炎病毒结构蛋白的表达，出现了类似人类急性病毒性肝炎的急性肝损伤。伴有肝细胞凋亡性死亡。转基因小鼠在注射AxCANCre后7d检测到血清核心蛋白，血清ALT明显升高，随后在感染后14d检测到抗核心抗体应答。此外，CD4和CD8阳性细胞耗竭试验使小鼠血清ALT升高和肝脏病理改变正常化。这些结果表明，丙型肝炎病毒蛋白不是直接的细胞病变，宿主免疫反应在丙型肝炎病毒感染中起着关键作用。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. We established HCV transgenic mouse model using Cre/loxP switching system. We put neomycin resistance gene as a stuffer flanked by loxP sequence at both 5' and 3' end between CAG promoter and core to NS2, HCV cDNA CN2. Spleens were harvested from CN2 transgenic mice. In 2 lineages out of 8, cultured spleen cell and fibroblast highly expressed core, E1 and E2 proteins by western blot and immunostaining after infection with recombinant adeno virus (AxCANCre) which express cre DNA recombinase in infected cell. To induce HCV expression in vivo and in the liver, we injected AxCANCre to CN2 transgenic mouse from tail vein and HCV protein expression was confirmed in its liver tissue. With the expression of HCV structural protein, acute liver injury which was similar to human acute viral hepatitis was occurred. It was accompanied with apoptotic liver cell death. Serum core protein was detected in transgenic mice 7 days after AxCANCre injection with evident serum ALT elevations, subsequently, anti-core antibody response was detected at 14 days after infection. Furthermore, CD4 and CD8 positive cell depletion assay normalized the serum ALT elevations of mice and pathological changes in the liver. These results suggest that HCV proteins are not direct cytopathic and the host immune response has a pivotal roles in HCV infection. 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.