The Pathogenesis of Steatosis in Hepatitis C Genotype 3 Infection

基因型 3 丙型肝炎感染脂肪变性的发病机制

• 批准号: 7463764
• 负责人: RAVI R. JHAVERI
• 金额: $ 12.43万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-09 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7463764
• 关键词: Adenovirus Infections; Amino Acid Substitution; Amino Acids; Antiviral Therapy; Biochemistry; Biology; Biopsy; Cell Line; Cells; Chronic; Clinical; Communicable Diseases; Core Protein; Counseling; DNA Sequencing Facility; Databases; Development; Elements; Fatty acid glycerol esters; Fibrosis; Flow Cytometry; Frequencies; Genes; Genetic Polymorphism; Genotype; Hand; Health; Hepatitis; Hepatitis C; Hepatitis C virus; Hepatocyte; Histology; In Vitro; Infection; Inpatients; Integration Host Factors; Intracellular Accumulation of Lipids; Laboratories; Lead; Light; Link; Lipids; Liver; Malignant Neoplasms; Mentors; Methods; Microscopy; Molecular Biology; Molecular Virology; Mus; Numbers; Pathogenesis; Patients; Physicians; Proteins; Research; Research Personnel; Rest; Reverse Transcriptase Polymerase Chain Reaction; Risk Factors; Sampling; Scientist; Sequence Analysis; Severities; Signal Transduction; Specimen; Statistical Methods; Testing; Training; Viral; Viral Genes; Work; base; carcinogenesis; cohort; experience; hepatitis C virus nucleocapsid protein; improved; insight; interest; intrahepatic; liver biopsy; microsomal triglyceride transfer protein; mutant; novel; programs; protein expression; response; viral RNA

DESCRIPTION (provided by applicant): This proposal will provide me with mentoring, hands on research experience and needed didactic training in order to develop into an independent physician-scientist. I am trained in Infectious Diseases and this proposal will advance my research interest in Hepatitis C virus (HCV). Steatosis, or fat accumulation within hepatocytes, is a major component of HCV-induced changes in liver histology. HCV genotype 3, unlike other genotypes, is an independent risk factor for both the presence and severity of steatosis. My preliminary sequence analysis of patient derived HCV RNA identified amino acid polymorphisms within Domain 3 of the Core protein that correlate with the presence or absence of steatosis. In vitro expression of these patient derived clones leads to intracellular fat accumulation. In this proposal, I will test the hypotheses that: 1) specific amino acid substitutions within Domain 3 of the Core protein are observed in patients with steatosis in HCV genotype 3 infection; 2) these amino acid substitutions are required to cause intracellular lipid accumulation when introduced into cultured hepatocyte cell lines; 3) intracellular fat in HCV infection contributes to fibrosis formation in the liver via interactions between HCV Core protein, Microsomal Triglyceride Transfer Protein (MTP), CD1d and NKT cells. In order to test the hypotheses listed above, I will use a combination of RT-PCR amplification of viral genes, in vitro expression, light and fluoresecent microscopy, recomibinant adenovirus infections in mice and flow cytometry. I will be using patient samples from a well characterized specimen bank from over 400 patients with HCV infection in a laboratory that focuses on basic liver biochemistry, molecular biology and intracellular signaling. I have assembled a team of mentors and consultants with expertise in liver biology and Hepatitis C clinical and molecular virology. We are examining differences between HCV genotype 3 isolates rather than between different genotypes. These stuides will provide a basis for studies of mechanisms whereby HCV infection causes intrahepatic fat accumulation that leads to fibrosis formation when no mechanism has previously been proposed. Delineation of the mechanisms underlying steatosis in HCV will lead to a greater insight into fibrosis and cancer formation. Results from this work may improve counselling of HCV patients with steatosis and may facilitate efforts to identify novel targets for anti-viral development.

描述(由申请人提供)： 这项提议将为我提供指导、实践研究经验和必要的教学培训，以便我发展成为一名独立的内科科学家。我受过传染病方面的培训，这项建议将提高我对丙型肝炎病毒(丙型肝炎病毒)的研究兴趣。脂肪变性，即肝细胞内的脂肪堆积，是丙型肝炎病毒引起的肝脏组织学改变的主要组成部分。与其他基因型不同，丙型肝炎病毒基因3型是脂肪变性存在和严重程度的独立危险因素。我对患者来源的丙型肝炎病毒RNA的初步序列分析发现，核心蛋白第3区域内的氨基酸多态与脂肪变性的存在或不存在相关。这些患者来源的克隆在体外表达会导致细胞内脂肪积聚。在这项提议中，我将测试假设：1)在丙型肝炎病毒3型感染的脂肪变性患者中，观察到核心蛋白结构域3内的特定氨基酸替换；2)当被引入培养的肝细胞系时，这些氨基酸替换是必需的，以引起细胞内脂肪堆积；3)丙型肝炎病毒感染中的细胞内脂肪通过丙型肝炎病毒核心蛋白、微粒体甘油三酯转移蛋白(MTP)、CD1d和NKT细胞之间的相互作用而促进肝脏纤维化的形成。为了验证上述假设，我将使用RT-PCR病毒基因扩增、体外表达、光学显微镜和荧光显微镜、小鼠重组腺病毒感染和流式细胞术相结合的方法。我将在一个专注于基本肝脏生物化学、分子生物学和细胞内信号的实验室里，使用来自400多名丙型肝炎病毒感染患者的精心设计的标本库中的患者样本。我已经组建了一支在肝脏生物学、丙型肝炎临床和分子病毒学方面具有专业知识的导师和顾问团队。我们正在检查丙型肝炎病毒3型分离株之间的差异，而不是不同基因型之间的差异。这些研究将为研究丙型肝炎病毒感染导致肝内脂肪堆积导致纤维化形成的机制提供基础，而此前尚未提出任何机制。描述丙型肝炎脂肪变性的潜在机制将导致对纤维化和癌症形成的更深入的了解。这项工作的结果可能会改善对患有脂肪变性的丙型肝炎患者的咨询，并可能有助于确定抗病毒开发的新靶点。