MOLECULAR PATHOPHYSIOLOGY OF ALCHOLIC LIVER DISEASE

目的是为了更好地了解分子 酒精对肝功能的影响以及 酒精性肝病肝纤维化 两大调查领域 将研究：（1）什么水平的基因调控，特别是 胶原基因调控在酒精性肝病中受到影响？ 这 将涉及通过无细胞蛋白分析mRNA稳态水平 合成和RNA-DNA杂交。 的定量测定 几种特定mRNA种类（包括I型、III型和IV型）的变化 前胶原蛋白、白蛋白、纤连蛋白和β-肌动蛋白将被进行。 核 将进行转录径流分析，以检查 观察到的稳态mRNA含量变化的基础。 胶原基因 启动子结构和功能将通过构建部分 从酗酒者和非酗酒者的基因组文库中，分离 纯化胶原蛋白启动子，用S1 核酸酶敏感性甲基化、DNA测序和使用瞬时 表达载体pSV-0-CAT。 后面的这些研究可能解释了 对酒精的纤维化反应 (2)哪些因素 容易导致酒精性肝硬化吗 这将涉及 使用DNA多态性对胶原基因进行遗传分析， 性激素和皮质类固醇在肝硬化中的作用分析 胶原蛋白合成用细胞培养技术，分子杂交 研究和体内瞬时表达载体分析。 的结果 这样的实验将帮助我们扩展我们的分子知识， 机制负责肝纤维化的人。这一知识将 有助于开发评估纤维化的诊断方法 预测发展为肝硬化的易感性，并为 更好的治疗 这些研究也将有助于我的长期目标 成为学术肝病学的独立研究者， 在分子生物学的当前领域。

The objectives are to obtain a better understanding of the molecular effects of alcohol upon hepatic function as well as the pathogenesis of hepatic fibrosis in alcoholic liver disease. Two general areas of inquiry will be investigated: (1) What levels of gene regulation and specifically collagen gene regulation are affected in alcoholic liver disease? This will involve an analysis of mRNA steady state levels by cell free protein synthesis and RNA-DNA hybridization. Quantitative determinations of changes in several specific mRNA species including types I, III, and IV procollagen, albumin, fibronectin and -actin will be undertaken. Nuclear transcriptional run-off analysis will be performed in order to examine the basis for observed changes in steady state mRNA contents. Collagen gene promoter structure and function will be determined by constructing partial genomic libraries from cirrhotics and non-cirrhotic alcoholics, isolating and purifying the collagen promoter, and analyzing it by means of S1 nuclease sensitivity methylation, DNA sequencing, and use of a transient expression vector (pSV-0-CAT). These latter studies may explain variations in an individual's fibrotic response to alcohol. (2) What factors predispose an individual to develop alcoholic cirrhosis? This will involve a genetic analysis of the collagen gene using DNA polymorphisms and analysis of the role of sex hormones and corticosteroids in hepatic collagen synthesis using cell culture techniques, molecular hybridization studies and in vivo transient expression vector analysis. The results of such experiments will help us expand our knowledge of the molecular mechanisms responsible for hepatic fibrosis in man. This knowledge will help in developing diagnostic methods for evaluating fibrogenesis predicting susceptibility to develop cirrhosis, and provide the basis for better therapy. These studies will also facilitate my long term objective to become an independent investigator in academic hepatology with expertise in current areas of molecular biology.