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Analysis of viral counter regulation against host's defense mechanism for viral infection using HCV transgenic mouse model

利用HCV转基因小鼠模型分析病毒对宿主病毒感染防御机制的反调节

基本信息

批准号：
09470088
负责人：
WAKITA Takaji
金额：
$ 6.66万
依托单位：
Tokyo Metropolitan Institute for Neuroscience (1998)Tokyo Metropolitan Organization for Medical Research (1997)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09470088/
关键词：
HCV TRANSGENIC MOUSE Cre loxP CTL

项目摘要

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.

由于缺乏合适的培养体系和动物模型，阻碍了对病毒生活周期和HCV感染发病机制的深入研究。利用Cre/loxP转换系统建立了HCV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'端和3'端两侧插入loxP序列，插入NS 2，HCV cDNA CN 2。从CN 2转基因小鼠中收获脾脏。用表达cre DNA重组酶的重组腺病毒（AxCANCre）感染培养的脾细胞和成纤维细胞后，Western blot和免疫组化染色显示，在8个谱系中有2个谱系的脾细胞和成纤维细胞高表达core、E1和E2蛋白。为了诱导HCV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，并在其肝脏组织中证实了HCV蛋白表达。随着HCV结构蛋白的表达，出现了类似于人类急性病毒性肝炎的急性肝损伤。同时伴有肝细胞凋亡。转基因小鼠在AxCANCre注射后7天检测到血清核心蛋白，血清ALT明显升高，随后在感染后14天检测到抗核心抗体应答。此外，CD 4和CD 8阳性细胞耗竭试验使小鼠血清ALT升高和肝脏病理变化正常化。这些结果表明，HCV蛋白不是直接致细胞病变的，宿主免疫应答在HCV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HCV感染和HCV肝细胞癌变中的作用。