生成一株支持拉米夫定耐药乙型肝炎病毒有效复制的人肝癌细胞系。

Generation of a human hepatoma cell line supporting efficient replication of a lamivudine resistant hepatitis B virus.

机构信息

Key Laboratory of Medical Molecular Virology of the Ministries of Education and Health and Department of Infectious Disease, Huashan Hospital, Fudan University, Shanghai 200040, China.

Key Laboratory of Infection and Oncology, Research Center of Molecular Medicine, Fujian Medical University, Fuzhou, China.

出版信息

J Virol Methods. 2014 Jun;201:51-6. doi: 10.1016/j.jviromet.2014.02.008. Epub 2014 Feb 28.

DOI:10.1016/j.jviromet.2014.02.008

PMID:24583110

Abstract

Emergence of lamivudine (LAM) resistance causes treatment failure in patients with chronic hepatitis B and compromise the efficacy of subsequent salvage therapies with other nucleot(s)ide analogs (NAs). Establishment of cell-based assays supporting LAM-resistant hepatitis B virus (HBV) replication will not only provide tools for investigating the replication property, but also screening for antiviral agents efficiently inhibiting the replication of LAM-resistant HBV variants. Accordingly, a human hepatoma (HepG2)-derived cell line was established by stable transfection of a plasmid containing a 1.2 unit length of HBV genome harboring rtL180M and rtM204V mutations that confer LAM resistance. In addition to support efficient viral genome replication, the cell line also produces high levels of HBV virions and subviral particles. As expected, HBV DNA replication in this cell line is completely resistant to lamivudine, but sensitive to adefovir (ADV), entecavir (ETV) and tenofovir (TDF). The cell line is suitable for screening for antiviral agents that inhibit LAM-resistant HBV replication and inhibitors of HBsAg biosynthesis and secretion, which may reduce HBsAg antigenemia and ultimately help to restore host antiviral immune response against HBV and cure chronic HBV infection.

摘要

拉米夫定（LAM）耐药的出现导致慢性乙型肝炎患者的治疗失败，并影响后续使用其他核苷（酸）类似物（NAs）进行挽救治疗的疗效。建立支持拉米夫定耐药乙型肝炎病毒（HBV）复制的基于细胞的检测方法，不仅将为研究复制特性提供工具，还将有效筛选抑制拉米夫定耐药 HBV 变异体复制的抗病毒药物。因此，通过稳定转染含有 rtL180M 和 rtM204V 突变的 1.2 个单位长度 HBV 基因组的质粒，建立了人肝癌（HepG2）衍生细胞系，该突变赋予了拉米夫定耐药性。除了支持高效的病毒基因组复制外，该细胞系还产生高水平的 HBV 病毒颗粒和亚病毒颗粒。正如预期的那样，该细胞系中的 HBV DNA 复制完全对拉米夫定耐药，但对阿德福韦酯（ADV）、恩替卡韦（ETV）和替诺福韦（TDF）敏感。该细胞系适合筛选抑制拉米夫定耐药 HBV 复制的抗病毒药物以及抑制 HBsAg 生物合成和分泌的抑制剂，这可能会降低 HBsAg 抗原血症，并最终有助于恢复宿主对乙型肝炎病毒的抗病毒免疫反应并治愈慢性乙型肝炎感染。