HIV replication and cell injury in cultured hepatocytes

培养肝细胞中的 HIV 复制和细胞损伤

• 批准号: 6501009
• 负责人: NELSON FAUSTO
• 金额: $ 21.22万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6501009
• 关键词: CD95 molecule; apoptosis; cooperative study; cytokine; embryo /fetus tissue /cell culture; hepatitis C; hepatitis C virus; liver cells; terminal nick end labeling; tissue /cell culture; transfection; virus RNA; virus cytopathogenic effect; virus replication

Description (adapted from the application). The lack of an adequate cell culture system to study HCV replication has been a major obstacle to progress in understanding the pathogenesis of hepatitis C. The goal of this laboratory has been to establish cultures of non-transformed human hepatocyte, which retain the expression of liver-specific markers and are suitable for the study of interactions between HCV and its natural host, the normal human hepatocyte. This project describes the general features of a newly established culture system using cultured human fetal hepatocytes (HFH). Cells transfected with full length HCV RNA (WT) but not with a 3'delta-deletion mutant, express core protein and show virus replication as demonstrated by strand-specific in situ hybridization (ISH). In cultures transfected with WT HCV RNA but not in those transfected with mutant RNA, cells are vacuolated, grow in a nodular pattern and apoptosis demonstrated by TUNEL or 'live dead' assays is evident. It is proposed that apoptotic elimination of sensitive cells, preservation of permissive cells in which the virus persists and activation of cytokine mediated viral clearance mechanisms are events that occur during acute HCV infection and set the stage for the outcome of the infection. Experiments have been designed to test these hypotheses and to determine in clinical samples whether specific mutations and quasispecies evolution may correlate with HCV replication and cytopathic effects in cultured human hepatocytes. Experiments described in Aim 1 will determine the main parameters of viral replication and infectivity in HFH cultures transfected with HCV RNA or infected with patient sera and explore new methods to increase the efficiency of transfection or infection. Aim 2 will examine the expression of apoptotic markers in HFH cultures and investigate whether oxidant damage, Fas-Fas-L expression and cytokine-mediated signaling pathways are important mechanisms in HCV-induced hepatocyte apoptosis. Project 1 is directly linked to Projects 2 and 3 and Core A. The cDNA microarray gene expression analysis described in Project 2 will provide a comprehensive picture of the HFH culture system, HCV cytopathic effects and IFN signaling. Clinical samples obtained in studies described in Project 3 and prepared by Core A will be used to test the hypothesis that clinical isolates well characterized as to genotype, quasispecies and disease outcome in patients will differ in their capacity to produce cytopathic effects in hepatocytes.

描述(改编自应用程序)。缺乏足够的牢房 研究丙型肝炎病毒复制的培养体系一直是进展的主要障碍 为了了解丙型肝炎的发病机制，这个实验室的目标是 已经建立了未转化的人肝细胞的培养，这 保留肝脏特异性标志物的表达，适合于研究 研究丙型肝炎病毒与其自然宿主--正常人类肝细胞之间的相互作用。 这个项目描述了一种新建立的文化的总体特征。 体外培养人胎肝细胞(HFH)。转染成纤维细胞 全长丙型肝炎病毒RNA(WT)，但不含3‘端增量缺失突变体，EXPRESS核心 蛋白质和显示病毒复制的链特异性原位研究 杂交(ISH)。在感染了WT-HCVRNA的培养上，而在那些 携带突变核糖核酸的细胞空泡化，呈结节状生长 原位末端标记法或“活体死亡”试验显示细胞凋亡明显。它是 建议消除敏感细胞的凋亡，保存 病毒持续存在的允许细胞和细胞因子的激活 介导的病毒清除机制是在急性丙型肝炎病毒感染期间发生的事件 并为感染的结果做好准备。实验 已经被设计用来测试这些假说并在临床上确定 样本：特定突变和准物种进化是否相关 在培养的人肝细胞中具有丙型肝炎病毒复制和细胞病变效应。 目标1中描述的实验将确定病毒的主要参数 丙型肝炎病毒核糖核酸和丙型肝炎病毒在HFH细胞中的复制和感染性 感染患者血清，探索提高效率的新方法 关于转基因或感染的。AIM 2将检测细胞凋亡的表达 标志，并调查氧化剂是否损伤，Fas-Fas-L 表达和细胞因子介导的信号通路是重要的机制 在丙型肝炎病毒诱导的肝细胞凋亡中。项目%1直接链接到项目 2和3和Core A.中描述的基因微阵列基因表达分析 项目2将全面介绍HFH培养系统--丙型肝炎病毒 细胞病变效应与干扰素信号转导。研究中获得的临床样本 项目3中描述的并由Core A准备的将用于测试 假设临床分离株具有很好的基因型特征， 患者的准种和疾病结局将在他们的能力上有所不同 在肝细胞中产生细胞病变效应。