Novel Robust in Vitro Hepatitis B Virus Infection Model Using Fresh Human Hepatocytes Isolated from Humanized Mice
Novel Robust in Vitro Hepatitis B Virus Infection Model Using Fresh Human Hepatocytes Isolated from Humanized Mice
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DOI：
10.1016/j.ajpath.2015.01.028
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发表时间：
2015-05-01
影响因子：
6
通讯作者：
Tateno, Chise
中科院分区：
文献类型：
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作者：
Ishida, Yuji;Yamasaki, Chihiro;Tateno, Chise
由于缺乏适当的体外感染模型，乙型肝炎病毒（HBV）生命周期的分子机制尚不清楚。在此，我们报道了一个高效的体外HBV感染系统，使用从人源化肝脏嵌合小鼠中分离的新鲜人肝细胞（HHs）。将HBV感染嵌合小鼠或患者的血清接种HHs后，我们测量了HHs中HBV DNA、mRNA、共价闭合环状DNA和病毒蛋白的表达水平。研究了乙型肝炎免疫球蛋白的中和活性以及siRNA对牛磺胆酸钠共转运多肽和网状蛋白重链对HBV感染的影响。我们证实了HHs中病毒抗原的表达以及细胞外HBV DNA和乙型肝炎表面抗原的存在。最高感染率约为80%。拉米夫定和乙肝免疫球蛋白治疗以剂量依赖的方式降低HBV DNA水平。敲低牛磺酸胆酸钠共转运多肽和网状蛋白重链可显著降低乙型肝炎表面抗原水平。使用不同的供体HHs和接种疫苗成功建立感染。持续的乙肝免疫球蛋白治疗可阻断细胞外HBV DNA水平的升高和HBV阳性HHs的增加，提示病毒在该模型中传播。嵌合小鼠衍生的HHs提供了一个强大的体外感染模型，可以完全支持HBV的生命周期。
The molecular mechanisms underlying the hepatitis B virus (HBV) Life cycle are poorly understood because of the lack of appropriate in vitro infection models. Herein, we report a highly effective in vitro HBV infection system using fresh human hepatocytes (HHs) isolated from chimeric mice with humanized livers. After the inoculation of sera collected from HBV-infected chimeric mice or patients to HHs, we measured levels of HBV DNA, mRNA, covalently closed circular DNA, and viral protein expression in HHs. We investigated the neutralization activity of hepatitis B immune globulin and the effects of siRNA against sodium taurocholate cotransporting polypeptide and clathrin heavy chain on HBV infection. We confirmed the expression of viral antigens in HHs and the presence of extracellular HBV DNA and hepatitis B surface antigen. The maximum infection rate was approximately 80%. Lamivudine and hepatitis B immune globulin treatment reduced HBV DNA levels in a dose-dependent manner. Knockdown of sodium taurocholate cotransporting polypeptide and clathrin heavy chain significantly reduced the levels of hepatitis B surface antigen. Infection was successfully established using different donor HHs and inocula. Elevation of extracellular HBV DNA levels and the increase of HBV-positive HHs were blocked by continuous hepatitis B immune globulin treatment, indicating virus spread in this model. Chimeric mouse derived HHs provide a robust in vitro infection model that can completely support the HBV life cycle.