Production of Infectious Hepatitis C Virus in Primary Cultures of Human Adult Hepatocytes
Production of Infectious Hepatitis C Virus in Primary Cultures of Human Adult Hepatocytes
复制标题
DOI：
10.1053/j.gastro.2010.06.058
复制
发表时间：
2010-10-01
期刊：
影响因子：
29.4
通讯作者：
Calmus, Yvon
中科院分区：
文献类型：
--
作者：
Podevin, Philippe;Carpentier, Arnaud;Calmus, Yvon
背景与目的：虽然丙型肝炎病毒（HCV）可以在肝癌衍生的细胞系Huh-7中生长，但需要一种细胞培养模型来支持其在正常、静止、高度分化的人肝细胞中的完整、生产性感染周期。我们试图建立这样一个系统。方法：将人成年肝细胞的原代培养物接种来源于Huh-7细胞培养物（HCVDNA）的HCV，并监测肝细胞分化标志物的表达和HCV的复制。测定培养物上清液的HCV RNA、核心抗原和感染性滴度。在碘克沙醇梯度中比较输入和子代病毒的浮力密度。研究结果：原代肝细胞在保持分化标志物表达的同时，支持HCV的完整感染循环，包括产生显著滴度的新感染性子代病毒，其被称为原代培养物衍生病毒（HCVpc）。与HCVpc相比，HCVpc具有较低的平均浮力密度和较高的特异性感染性;这与体内感染过程中产生的极低密度脂蛋白相关的病毒颗粒的特征相似。在低分化的Huh-7细胞中再培养HCVpc后，这些特性丢失，这表明真正的病毒体只能由分泌真正的极低密度脂蛋白的正常肝细胞产生。结论：我们已经建立了一个基于细胞培养的系统，允许在生理相关的人肝细胞中生产感染性HCV。这为研究HCV与其天然宿主细胞的相互作用以及开发抗病毒治疗提供了有用的工具。
BACKGROUND & AIMS: Although hepatitis C virus (HCV) can be grown in the hepatocarcinoma-derived cell line Huh-7, a cell-culture model is needed that supports its complete, productive infection cycle in normal, quiescent, highly differentiated human hepatocytes. We sought to develop such a system. METHODS: Primary cultures of human adult hepatocytes were inoculated with HCV derived from Huh-7 cell culture (HCVcc) and monitored for expression of hepatocyte differentiation markers and replication of HCV. Culture supernatants were assayed for HCV RNA, core antigen, and infectivity titer. The buoyant densities of input and progeny virus were compared in iodixanol gradients. RESULTS: While retaining expression of differentiation markers, primary hepatocytes supported the complete infectious cycle of HCV, including production of significant titers of new infectious progeny virus, which was called primary-culture-derived virus (HCVpc). Compared with HCVcc, HCVpc had lower average buoyant density and higher specific infectivity; this was similar to the characteristics of virus particles associated with the very-low-density lipoproteins that are produced during in vivo infection. These properties were lost after re-culture of HCVpc in poorly differentiated Huh-7 cells, suggesting that authentic virions can be produced only by normal hepatocytes that secrete authentic very-low-density lipoproteins. CONCLUSIONS: We have established a cell-culture-based system that allows production of infectious HCV in physiologically relevant human hepatocytes. This provides a useful tool for the study of HCV interactions with its natural host cell and for the development of antiviral therapies.