HBV DNA转染原代鸭肝细胞初步观察

目的：通过观测人HBV DNA在非哺乳动物－－鸭肝细胞中的复制和表达水平，探讨人HBV感染与复制的跨种属特异性，为建立人HBV DNA转染跨种属肝细胞模型奠定基础。方法：获取线性HBV DNA并电转染原代鸭肝细胞，电转后48h采用IMX系统检测鸭肝细胞中HBsAg表达水平，用Southern blot-ting和dot blotting检测HBV DNA复制情况。以单纯电击肝细胞为对照。结果：转染组原代鸭肝细胞裂解液中HBsAg为9．10（P／N值≥2．1为阳性），HBeAg为阴性；上清液中二者均为阴性。转染组原代鸭肝细胞裂解液dot blotting呈强阳性；转染组肝细胞总DNA Southern blotting显示约4.0kb以下分子涂抹带，为游离复制型HBV DNA，包括rcDNA,cccDNA与ssDNA等复制中间体，未见整合型HBV DNA－－高分子区（4.0-24.0kb)涂抹带，对照上述指标均为阴性。结论：人HBV DNA能在原代鸭肝细胞中复制和表达，可能为肝细胞内环境依赖性，无严格种属特异性限制。

Replication and transfection of hepatitis B virus DNA into primary duck hepatocytes

OBJECTIVE: By studying the possibility of obtaining expression of human hepatitis B virus (HBV) genes and production in normal liver cells from heterologous species like normal primary duck hepatocytes (PDH), to investigate the species-specificity of HBV infection and replication. METHODS: Two days after transfecting the complete HBV genome into PDH by electroporation (transfected group), HBsAg and HBeAg in the supernatants and lysates of PDH were measured by the IMX system. Meanwhile, replication of HBV in PDH was analyzed by Southern blotting and dot blotting procedures. PDH was electroporated as control. RESULTS: HBsAg in the lysate of transfected group was 9.10 (P/N values, positive?2.1), HBeAg was 1.0 (negative?2.1), both were negative in the supernatants of transfected group. dot blotting revealed that transfected group was strongly positive, whereas the control group was negative. Southern blot analysis of intracellular total DNA indicated that there were relaxed circular (RC), covalently closed circular (CCC) and single-stranded (SS) HBV DNA replicative intermediates in the transfected group, and there was no integrated HBV DNA in the cellular genome. Control groups were negative. CONCLUSIONS: Replication of HBV can occur in hepatocytes from nonmammalian species, which strongly supports the idea that replication of HBV has no critical species-specificity, and yet it depends on the endoenvironment of hepatocyte.