Studies on proliferating mechanism of hepatitis C virus using cultured human liver cell lines by radialflowtype-bioreactor

为了构建感染人工肝的感染性克隆，我们从一名丙型肝炎病毒携带者的血液样本中构建了丙型肝炎病毒的全长cDNA克隆。血液样本被证明对接受者和黑猩猩都有传染性。然后利用Charomid系统构建了含丙型肝炎病毒全长c DNA的重组杆状病毒。通过免疫沉淀分析，在感染重组杆状病毒的细胞中表达经过适当处理的丙型肝炎病毒蛋白，然后评估它们在人工丙型肝炎病毒感染和复制模型中的适用性。我们构建了CAG启动子(AdexCAT7)控制下表达噬菌体T7 RNA聚合酶的复制缺陷重组腺病毒。可检测到高水平的T7RNA聚合酶。然后将携带T7启动子、脑心肌炎病毒5‘非翻译区和…的载体导入感染AdexCAT7的细胞更多的是荧光素酶(PT7EMCVLuc)，或(Ii)T7启动子，丙型肝炎病毒的5‘非编码区和一个荧光素酶基因(PT7HCVLuc)。PT7EMCVLuc与p7F7HCVLuc相比，多数细胞系支持荧光素酶的高表达。然而，一株来源于人肝细胞癌的细胞系FLC4显示出pT7HCVLuc报告基因的高表达。在该细胞系中，体外合成的pT7HCVLuc的RNA比pT7EMCVLuc的RNA诱导了更高水平的报告基因表达。这可能是由于一些未知的因素使HCVRNA在FLC4中的微小基因转录过程具有很强的稳定性。在单层培养中，当用一例慢性丙型肝炎患者的血浆进行实验时，FLC4细胞表现出持续的丙型肝炎病毒RNA分泌，这些发现说服我们选择FLC4细胞作为最适合于进一步研究丙型肝炎病毒感染和复制的人工模型。成功地在生物反应器环境中进行了稳定的高密度培养60d以上，而且通过低温培养可以控制增殖速度。使用该人工肝模型，可以通过RT-PCR确认丙型肝炎病毒感染后前两天血浆丙型肝炎病毒RNA的分泌，但在感染后的前两天检测为阴性。不幸的是，培养液的感染还不能得到证实。我们目前正在继续使用我们开发的感染性克隆以及从美国国立卫生研究院获得的已被证明感染黑猩猩的克隆进行研究。较少

In order to carry out a construction of infectious clones to infect into the artificial bio-liver, we constructed a full length cDNA clone of hepatitis C virus (HCV) from a blood sample of an HCV carrier. The blood sample was shown to be infectious to both a recipient and chimpanzees. Then we constructed a recombinant baculovirus containing the full length cDNA of HCV by using a charomid system. By immunoprecipitation analysis, properly processed HCV proteins were expressed in cells infected with the recombinant baculovirus.Cell lines were then assessed for their suitability in artificial models of HCV infection and replication. We constructed a replication-deficient recombinant adenovirus expressing bacteriophage T7 RNA polymerase under the control of CAG promoter (AdexCAT7). A high level of T7 RNA polymerase was detectable. Cells infected with AdexCAT7 were then transfected with plasmids carrying (i) the T7 promoter, the 5' untranslated region (UTR) of encephalomyocarditis virus and … More a luciferase (pT7EMCVLuc), or (ii) the T7 promoter, the 5'UTR of HCV and a luciferase gene (pT7HCVLuc). Most of the cell lines examined supported a higher expression of luciferase by transfection with pT7EMCVLuc than with p7F7HCVLuc. However, one cell line, FLC4, derived from a human hepatocellular carcinoma exhibited very high reporter gene expression with pT7HCVLuc. In this cell line, transfection with RNA synthesized in vitro from pT7HCVLuc induced a higher level of reporter gene expression than RNA from pT7EMCVLuc. It may be due to robust stability of the HCV RNA minigene transcription process in FLC4 owing to some as yet unknown factor. In monolayer culture, FLC4 cells showed continuous secretion of HCV RNA when experiments were carried out using plasma taken from a chronic Hepatitis C patient Taken together, these findings persuaded us to select FLC4 cells as the most suitable for further studies in artificial models of HCV infection and replication. Stable high-density cultivation in bioreactor environments was successfully carried out for more than 60 days, and furthermore, proliferation rates could be controlled by cultivating at lower temperatures.Using this artificial liver model, it was possible to confirm by RT-PCR the secretion of HCV RNA in response to Hepatitis C infected plasma HCV RNA tested positive for the first two days after infection, but tests were negative thereafter. Unfortunately, infection of the culture medium could not be confirmed.We are presently continuing studies using the infectious clones we have developed as well as those obtained from the NIH USA which have been shown to infect chimpanzees. Less