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[乙型肝炎病毒在异种原代肝细胞中复制的研究]

[Study on the replication of Hepatitis B Virus in primary hepatocytes from heterogenous species].

作者信息

Yao Yun-qing, Zhang Ding-feng, Huang Ai-long, Tang Ni, Wang Bo, Zhou Wei-ping, Ren Hong, Guo Shu-hua

机构信息

Department of Infectious Diseases, First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China.

出版信息

Zhonghua Gan Zang Bing Za Zhi. 2004 Jan;12(1):25-8.

Abstract

OBJECTIVES

By studying the possibility and degree of the replication and expression of human hepatitis B virus (HBV) genes in normal liver cells from heterogenous species, such as primary duck hepatocytes (PDH) and primary rat hepatocytes (PRH), to investigate the species-specificity of HBV infection and replication.

METHODS

PDH and PRH isolated by in situ perfusion with low concentration collagenase were transfected with complete HBV genome by electroporation (transfection group, about 1.19 10(12) copies of linear HBV DNA/1 10(7) PRH/PDH). From 1 day to 15 days after transfection, HBsAg and HBeAg in the supernatants and lysates of PRH/PDH were measured with IMX System, HBcAg was assayed with western blotting, Immunol dot blotting and Immunocytochemistry. Moreover, HBV S-mRNA and X-mRNA were tested with RT-PCR. Meanwhile, replicative intermediates of HBV DNA were analyzed by Southern blotting and Dot blotting. PRH/PDH electroporated only was used as control group.

RESULTS

HBsAg in the lysates of transfected PDH group were 15.24 (1day), 14.55 (3 days) and 5.13 ( 5 days; P/N values, positive>or=2.1), HBeAg all was negative (<2.1), and both were negative in the supernatants of transfected group. Viral antigen production in transfected rat hepatocytes: HBsAg in the lysates of transfected hepatocytes was positive, P/N values ranging from 2.17 to 93.41, The average P/N values was 14.74+/-31.82, and could be maintained for 15 days after transfection. Whereas, HBsAg in the supernatants of transfected group was only found positive on 1 day after transfection, which P/N value was 6.66. HBeAg and HBcAg in the lysates of PRH of transfection group were positive during the first 3 days following transfection, P/N values was around 2.8. The total amount of HBV DNA in the transfected PDH and PRH groups was strongly positive by dot blotting, whereas that of the control group was negative. Southern blot analysis of intracellular total HBV DNA indicated that there were relaxed circular (RC), covalently closed circular (ccc) and single-stranded (SS) HBV DNA replicative intermediates in the transfected PDH and PRH groups, there was no integrated HBV DNA in the cellular genome. Control groups were negative at all.

CONCLUSION

Expression of HBV genes and production can occur in hepatocytes from nonmammalian species or mammalian species, which strongly supports the idea that replication of HBV has no critical species-specificity, and yet it depends on the endoenvironment of hepatocyte.

摘要

目的

通过研究人类乙型肝炎病毒(HBV)基因在异种正常肝细胞(如原代鸭肝细胞(PDH)和原代大鼠肝细胞(PRH))中复制和表达的可能性及程度,探讨HBV感染和复制的种属特异性。

方法

采用低浓度胶原酶原位灌注法分离得到的PDH和PRH,通过电穿孔法用完整的HBV基因组进行转染(转染组,约1.19×10¹²拷贝线性HBV DNA/1×10⁷PRH/PDH)。转染后1天至15天,用IMX系统检测PRH/PDH上清液和裂解物中的HBsAg和HBeAg,用蛋白质印迹法、免疫斑点印迹法和免疫细胞化学法检测HBcAg。此外,用逆转录聚合酶链反应(RT-PCR)检测HBV S-mRNA和X-mRNA。同时,通过Southern印迹法和斑点印迹法分析HBV DNA的复制中间体。仅进行电穿孔的PRH/PDH作为对照组。

结果

转染的PDH组裂解物中的HBsAg分别为15.24(1天)、14.55(3天)和5.13(5天;P/N值,阳性≥2.1),HBeAg均为阴性(<2.1),转染组上清液中的两者均为阴性。转染大鼠肝细胞中的病毒抗原产生:转染肝细胞裂解物中的HBsAg呈阳性,P/N值在2.17至93.41之间,平均P/N值为14.74±31.82,转染后可维持15天。而转染组上清液中的HBsAg仅在转染后1天呈阳性,P/N值为6.66。转染组PRH裂解物中的HBeAg和HBcAg在转染后的前3天呈阳性,P/N值约为2.8。转染的PDH和PRH组中HBV DNA总量经斑点印迹法检测呈强阳性,而对照组为阴性。细胞内总HBV DNA的Southern印迹分析表明,转染的PDH和PRH组中存在松弛环状(RC)、共价闭合环状(ccc)和单链(SS)HBV DNA复制中间体,细胞基因组中无整合的HBV DNA。对照组均为阴性。

结论

HBV基因可在非哺乳动物或哺乳动物的肝细胞中表达和产生,这有力地支持了HBV复制无严格种属特异性的观点,但它取决于肝细胞的内环境。

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