Gong Z J, De Meyer S, Roskams T, van Pelt J F, Soumillion A, Crabbé T, Yap S H
Department of Liver and Pancreatic Diseases, University Hospital Gasthuisberg, Katholieke Universiteit Leuven, Belgium.
J Viral Hepat. 1998 Nov;5(6):377-87. doi: 10.1046/j.1365-2893.1998.00125.x.
Studies on the pathobiology of chronic (long-term) hepatitis B virus (HBV) infection and in vitro drug testing have been hampered by the lack of appropriate systems for culturing susceptible cells chronically infected with HBV. Most of the in vitro studies of HBV replication have been performed with HBV genome-transduced cell lines. In this system, viral production is mainly the result of chromosomal replication. In an in vitro infection system, owing to medium refreshment (which leads to the removal of infectious particles necessary for the perpetuation of infection) and to trypsinization for cell passages, it is difficult, if not impossible, to maintain chronic HBV infection, despite the use of susceptible cells. To circumvent these unfavourable factors for chronic HBV infection in vitro, we cultured microcarrier-attached immortalized human hepatocytes, infected with HBV, in molecularporous (MW 12,000-14,000) membrane (dialysis) bags for a duration of 2 months. HBV covalently-closed-circular (ccc) DNA, HBV precore/core and X mRNAs were detected in the cells cultured in this system following infection until the end of the experiment (day 58), while in classical culture conditions (monolayer), markers of HBV replication were also detected. Production of hepatitis B surface antigen (HBsAg) and HBV DNA was detected and their levels in culture medium (collected at the end of experiments from the molecularporous membrane bags) were increased 2.86- and 3.28-fold respectively. Using Southern blot analysis, HBV replicative intermediates could also be demonstrated throughout the experiments. However, integrated HBV DNA was not present. In contrast, HBV ccc DNA, HBV precore/core and X mRNAs, and replicative intermediates were not demonstrable in FTO 2B rat hepatoma cells infected in the same manner in parallel experiments. This in vitro infection system, using susceptible, immortalized human hepatocytes, therefore provides a new tool for studying the long-term effect of HBV infection, mainly involving episomal replication in hepatocytes, and for drug testing.
慢性（长期）乙型肝炎病毒（HBV）感染的病理生物学研究以及体外药物测试一直受到缺乏用于培养长期感染HBV的易感细胞的合适系统的阻碍。大多数HBV复制的体外研究是在HBV基因组转导的细胞系中进行的。在这个系统中，病毒产生主要是染色体复制的结果。在体外感染系统中，由于培养基更换（这会导致去除感染持续存在所需的感染性颗粒）以及为细胞传代进行胰蛋白酶消化，即使使用易感细胞，也很难（如果不是不可能的话）维持慢性HBV感染。为了规避体外慢性HBV感染的这些不利因素，我们将感染了HBV的微载体附着的永生化人肝细胞培养在分子多孔（分子量12,000 - 14,000）膜（透析）袋中长达2个月。在感染后直至实验结束（第58天），在该系统中培养的细胞中检测到了HBV共价闭合环状（ccc）DNA、HBV前核心/核心和X mRNA，而在经典培养条件（单层培养）下，也检测到了HBV复制的标志物。检测到了乙型肝炎表面抗原（HBsAg）和HBV DNA的产生，并且它们在培养基（在实验结束时从分子多孔膜袋中收集）中的水平分别增加了2.86倍和3.28倍。使用Southern印迹分析，在整个实验过程中也可以证明HBV复制中间体的存在。然而，不存在整合的HBV DNA。相比之下，在平行实验中以相同方式感染的FTO 2B大鼠肝癌细胞中，未检测到HBV ccc DNA、HBV前核心/核心和X mRNA以及复制中间体。因此，这种使用易感的、永生化人肝细胞的体外感染系统为研究HBV感染的长期影响（主要涉及肝细胞中的游离型复制）以及药物测试提供了一种新工具。
