摘要
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和 ＨＢｅＡｇ表达水平，用 Ｓｏｕｔｈｅｒｎ ｂｌｏｔ－ｔｉｎｇ和ｄｏｔ ｂ１ｏｔｔｉｎｇ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ｄｏｔｂｌｏｔｔｉｎｇ呈强阳性；转染组肝细胞总 ＤＮＡ Ｓｏｕｔｈｅｒｎ ｂｌｏｔｔｉｎｇ显示约４．０ ｋｂ以下分子涂抹带，为游离复制型 ＨＢＶＤＮＡ，包括 ｒｃＤＮＡ、ｃｃｃＤＮＡ与 ｓｓＤＮＡ等复制中间体；未见整合型 ＨＢＶ ＤＮＡ──高分子区（４．０－２４．０ ｋｂ）涂抹带。对照组上述指标均为阴性。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。
Objective By studying the possibility of obtaining expression of human hepatitis B virus (HIBV) 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 HIBV 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.
出处
《中华肝脏病杂志》
CAS
CSCD
2002年第1期34-36,共3页
Chinese Journal of Hepatology
基金
国家自然科学基金(39670340)
