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: 6659984
• 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: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6659984
• 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）。缺乏足够的细胞 研究HCV复制的培养系统一直是进展的主要障碍 丙型肝炎的发病机制。 这个实验室的目标 已经建立了非转化的人肝细胞的培养物， 保留肝脏特异性标志物的表达并适合于研究 丙型肝炎病毒与其自然宿主正常人肝细胞之间的相互作用。 本专题描述了一个新建立的文化的一般特征 使用培养的人胎肝细胞（HFH）的系统。 转染的细胞 全长HCV RNA（WT），但不含3 '缺失突变体，表达核心 蛋白质，并显示病毒复制，如链特异性原位 杂交（ISH）。 在用WT HCV RNA转染的培养物中， 用突变体RNA转染，细胞空泡化，以结节状模式生长， 并且通过TUNEL或“活死”测定证明的细胞凋亡是明显的。是 提出，敏感细胞的凋亡消除， 病毒持续存在的容许细胞和细胞因子的激活 介导的病毒清除机制是在急性HCV感染期间发生的事件， 感染并为感染的结果奠定基础。 实验 已经被设计来测试这些假设，并在临床上确定 样本是否特定突变和准种进化可能相关 在培养的人肝细胞中HCV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。 在用HCV RNA转染的HFH培养物中的复制和感染性， 感染患者血清，并探索新的方法，以提高效率 转染或感染。 目的2检测凋亡细胞的表达， 在HFH培养物中的标志物，并研究氧化损伤，Fas-Fas-L 表达和精氨酸介导的信号通路是重要的机制 HCV诱导的肝细胞凋亡。 项目1直接链接到项目 2和3以及核心A。 cDNA微阵列基因表达分析描述于 项目2将提供HFH培养系统、HCV 细胞病变效应和IFN信号传导。 研究中获得的临床样本 项目3中描述并由核心A准备的测试将用于测试 假设 充分表征的临床分离株 至于基因型， 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。