The Pathogenesis of Steatosis in Hepatitis C Genotype 3 Infection

描述（由申请人提供）： 这项建议将为我提供指导，实践研究经验和必要的教学培训，以便发展成为一名独立的医生科学家。我接受过传染病方面的培训，这项建议将促进我对丙型肝炎病毒（HCV）的研究兴趣。脂肪变性，或肝细胞内的脂肪积聚，是HCV诱导的肝组织学变化的主要组成部分。与其他基因型不同，HCV基因型3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HCV RNA进行的初步序列分析确定了核心蛋白结构域3内的氨基酸多态性，这些多态性与脂肪变性的存在或不存在相关。这些患者来源的克隆的体外表达导致细胞内脂肪积累。在这个提议中，我将检验以下假设：1）在HCV基因型3感染的脂肪变性患者中观察到核心蛋白的结构域3内的特定氨基酸取代; 2）当引入培养的肝细胞系时，这些氨基酸取代是引起细胞内脂质积聚所必需的; 3）HCV感染中的细胞内脂肪通过HCV核心蛋白、微粒体甘油三酯转移蛋白（MTP）、CD 1d和NKT细胞之间的相互作用而促成肝纤维化的形成。为了检验上述假设，我将使用病毒基因的RT-PCR扩增、体外表达、光学和荧光显微镜、重组腺病毒感染小鼠和流式细胞术的组合。我将使用来自一个实验室的400多名HCV感染患者的样本库的患者样本，该实验室专注于基础肝脏生物化学，分子生物学和细胞内信号传导。我已经组建了一个导师和顾问团队，他们在肝脏生物学和丙型肝炎临床和分子病毒学方面具有专业知识。我们正在研究HCV基因型3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HCV感染引起肝内脂肪堆积导致纤维化形成的机制研究提供基础，而以前没有提出任何机制。对HCV脂肪变性机制的阐明将使我们更深入地了解纤维化和癌症的形成。这项工作的结果可能会改善HCV脂肪变性患者的咨询，并可能有助于确定抗病毒开发的新靶点。

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.