Hepatitis C virus and liver fibrogenesis

丙型肝炎病毒与肝纤维化

基本信息

批准号：
7843580
负责人：
GULAM WARIS
金额：
$ 23.1万
依托单位：
ROSALIND FRANKLIN UNIV OF MEDICINE & SCI
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-05-15 至 2011-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7843580
关键词：
Adult Affinity Chromatography Amino Acids Antioxidants Calcium Signaling Cell Culture Techniques Cells Chelating Agents Chronic Chronic Hepatitis Chronic Hepatitis C Cirrhosis Cleaved cell Complex Cultured Cells Endoplasmic Reticulum Extracellular Matrix Fibrosis Figs - dietary Gene Expression Genome Genotype Goals Hepatic Fibrogenesis Hepatitis C Hepatitis C virus Hepatocyte Infection Injury Investigation Kinetics Link Liver Liver Fibrosis Liver diseases Malignant neoplasm of liver Measures Mediating Molecular Monitor Nonstructural Protein Oxidative Stress Pathway interactions Peptide Hydrolases Phosphotransferases Polyproteins Primary carcinoma of the liver cells Proteins RNA Reactive Oxygen Species Regulation Ribonucleoproteins Role Rough endoplasmic reticulum Signal Pathway Signal Transduction Small Interfering RNA Structural Protein System Testing Transforming Growth Factors Virus Virus Replication cytokine insight novel public health relevance response transcription factor viral RNA

项目摘要

DESCRIPTION (provided by applicant): The major goal of this proposal is to understand the molecular mechanism(s) of liver fibrosis by hepatitis C virus (HCV) infection. Hepatitis C virus (HCV) is the leading cause of chronic liver disease worldwide. HCV infection often leads to chronic hepatitis in up to 60-80% of infected adults and progresses to liver fibrosis, cirrhosis and hepatocellular carcinoma. The mechanisms underlying the liver injury and fibrosis in chronic hepatitis C are unclear. The HCV genome is a positive-sense single stranded RNA molecule that encodes a polyprotein precursor of ~3000 amino acids which is post- translationally cleaved by host and virus-encoded proteases into structural proteins (core, E1, and E2) and nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B). Recently, we have shown the elevated levels of ROS in cultured cells expressing HCV nonstructural proteins. This proposal's main hypothesis is that HCV-induced oxidative stress and Ca2+ signaling induce the synthesis and activation of transforming growth factor-21 (TGF-21) and its potential role in HCV replication and liver fibrosis. To test this hypothesis, the goal is to delineate the signaling pathways in induction of TGF-21 using recently described HCV cell culture infection system. The specific aims are to: 1) To define the kinetics of TGF-21 activation by different HCV genotypes (type 1b vs. 2a). 2) To analyze the role of HCV-induced Ca2+ signaling and oxidative stress in stimulating TGF-21 synthesis. 3) To define the role of cellular kinases and transcription factors on trans-regulation of TGF-21 gene expression. 4) To determine the effect of HCV-induced endogenous bioactive TGF-21 on HCV replication. The stimulation of TGF- 21 expression by HCV-induced oxidative stress and Ca2+ signaling, will be monitored by analyzing the RNA and protein levels of TGF- 21 from HCV infected cells and those treated with antioxidant and Ca2+ chelators. Next, TGF- 21 mediated regulation of HCV replication will be monitored by measuring the HCV RNA levels using quantitative RT- PCR. The assembly of HCV ribonucleoprotein complex (RNP) in response to TGF-21 will be examined by using two-step affinity purification of RNP complexes from HCV expressing cells silenced with TGF-21 siRNA. The proposed studies will yield novel insights into mechanisms of activation of profibrogenic factors that leads to hepatic fibrosis, cirrhosis and pave the way for delineating the pathways preceding liver cancer. PUBLIC HEALTH RELEVANCE: HCV infection often leads to chronic hepatitis which progresses to liver fibrosis, cirrhosis and hepatocellular carcinoma. HCV infected hepatocytes display oxidative stress which may provide a link between HCV infection and activation of TGF-21 that leads to progression of fibrosis. The information resulting from these investigations has a potential to provide clues to how HCV-induced oxidative stress and calcium signaling may induce profibrogenic factor, TGF-21, liver fibrosis and ultimately liver cancer.

描述（由申请人提供）：该提案的主要目标是了解丙型肝炎病毒（HCV）感染的肝纤维化分子机制。丙型肝炎病毒（HCV）是全球慢性肝病的主要原因。 HCV感染通常会导致高达60-80％的感染成年人的慢性肝炎，并发展为肝纤维化，肝硬化和肝细胞癌。慢性丙型肝炎中肝损伤和纤维化的基础机制尚不清楚。 The HCV genome is a positive-sense single stranded RNA molecule that encodes a polyprotein precursor of ~3000 amino acids which is post- translationally cleaved by host and virus-encoded proteases into structural proteins (core, E1, and E2) and nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B).最近，我们显示了表达HCV非结构蛋白的培养细胞中ROS的升高。该提案的主要假设是HCV诱导的氧化应激和Ca2+信号传导诱导转化生长因子21（TGF-21）的合成和激活及其在HCV复制和肝纤维化中的潜在作用。为了检验这一假设，目的是使用最近描述的HCV细胞培养感染系统来描述TGF-21诱导TGF-21的信号通路。具体目的是：1）定义不同HCV基因型（1B型与2A）激活TGF-21的动力学。 2）分析HCV诱导的Ca2+信号传导和氧化应激在刺激TGF-21合成中的作用。 3）定义细胞激酶和转录因子对TGF-21基因表达的反式调节的作用。 4）确定HCV诱导的内源性生物活性TGF-21对HCV复制的影响。 HCV诱导的氧化应激和Ca2+信号传导刺激TGF-21的表达，将通过分析来自HCV感染细胞的TGF-21的RNA和蛋白质水平以及用抗氧化剂和CA2+螯合剂治疗的RNA和蛋白质水平。接下来，将通过使用定量RT-PCR测量HCV RNA水平来监测TGF-21介导的HCV复制调节。通过使用来自HCV表达TGF-21 siRNA沉默的HCV的RNP复合物的两步亲和力纯化，将检查HCV核糖核蛋白复合物（RNP）对TGF-21的响应。拟议的研究将对肝纤维化，肝硬化，为描述肝癌前途径的途径铺平道路，对肝纤维化，肝硬化和铺平道路，从而产生新的见解。公共卫生相关性：HCV感染通常会导致慢性肝炎，该肝炎发展为肝纤维化，肝硬化和肝细胞癌。 HCV感染的肝细胞表现出氧化应激，这可能会提供HCV感染与TGF-21激活之间的联系，从而导致纤维化进展。这些研究产生的信息有可能提供有关HCV诱导的氧化应激和钙信号传导如何诱导纤维纤维成生因子TGF-21，肝纤维化和最终导致肝癌的线索。