GENE TECHNOLOGY THERAPY AND ALCOHOL-INDUCED FIBROSIS

基因技术治疗和酒精引起的纤维化

• 批准号: 6168539
• 负责人: RONALD G THURMAN
• 金额: $ 23.69万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-23 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6168539
• 关键词: alcoholism /alcohol abuse; biological models; cytokine receptors; fibrosis; gene therapy; genetically modified animals; inflammation; laboratory mouse; lipid metabolism; liver disorder; pathologic process; technology /technique development; transforming growth factors; tumor necrosis factor alpha

Pathologists have debated if fat or inflammation are necessary precursors of fibrosis for over a century. Importantly, we have very recently adapted the enteral alcohol delivery system developed for the rat by Tsukamoto and French to short-term studies in the mouse so that gene knockouts can be studied. Using the TNFR1 knockout in our new model, we obtained unequivocal evidence for the involvement of TNFalpha in early alcohol-induced liver injury. Mechanisms of alcohol-induced fibrosis remain less clear, and the purpose of this proposal is to use this new enteral model developed by us with knockout mice and gene delivery technqiues in long-term studies of alcohol- induced liver injury to fill important gaps in our knowledge. The unifying hypothesis we will test here using knockout technology is that inflammation but not fat is required for alcohol-induced fibrosis. We propose that this action is via oxidants. Initially, in Aim 1 we will optimize, characterize and validate a long-term mouse model of enteral alcohol delivery by determining pathology including fibrosis in wild-type mice which are background strains for knockouts selected for study. In Aim 2, we will take advantage of our pilot observation that the TNF receptor 1 knockout developed neither fat nor inflammation to test the hypothesis that fibrosis requires both. Next, we will study the effect of long-term enteral alcohol on fibrosis in knockouts which will exhibit fat but not inflammation (ICAM-1 knockouts) and inflammation but not fat (protein kinase A RIIbeta subunit knockouts). Finally, in Aim 3, we will use gene delivery of TGFbeta to TNF-R1 knockouts (i.e., no fat or inflammation) to test the hypothesis that alcohol-induced fibrosis is due to the pivotal cytokine TGFbeta alone. Next, antisense and dominant negatives to TGFbeta will be used to block fibrosis in wild-type mice. This work is timely and exciting since it will utilize our new enteral feeding model in the mouse and will allow us and others to investigate the roles of specific proteins and enzymes in alcohol-induced liver disease using the power of gene knockouts and gene delivery technology. This will position us uniquely to provide unequivocal new information and fill critical gaps in our knowledge on mechanisms of long-term alcohol-induced liver injury and set the stage for clinical trials.

一个多世纪以来，病理学家一直在争论脂肪或炎症是否是纤维化的必要先兆。 重要的是，我们最近将 Tsukamoto 和 French 为大鼠开发的肠内酒精输送系统应用于小鼠的短期研究，以便研究基因敲除。在我们的新模型中使用 TNFR1 敲除，我们获得了 TNFα 参与早期酒精性肝损伤的明确证据。 酒精引起的纤维化的机制尚不清楚，本提案的目的是利用我们用基因敲除小鼠开发的新肠内模型和基因递送技术对酒精引起的肝损伤进行长期研究，以填补我们的知识空白。我们将使用基因敲除技术在这里测试的统一假设是，酒精诱导的纤维化需要炎症而不是脂肪。 我们认为这种作用是通过氧化剂进行的。 最初，在目标 1 中，我们将通过确定野生型小鼠的病理学（包括纤维化）来优化、表征和验证肠内酒精输送的长期小鼠模型，野生型小鼠是选择用于研究的基因敲除的背景品系。 在目标 2 中，我们将利用我们的初步观察结果（即 TNF 受体 1 敲除既不产生脂肪也不产生炎症）来检验纤维化需要两者的假设。 接下来，我们将研究长期肠内酒精对表现出脂肪但不炎症（ICAM-1 敲除）和炎症但不脂肪（蛋白激酶 A RIIbeta 亚基敲除）的敲除纤维化的影响。 最后，在目标 3 中，我们将使用 TGFbeta 到 TNF-R1 敲除（即无脂肪或炎症）的基因传递来检验酒精诱导的纤维化仅由关键细胞因子 TGFbeta 引起的假设。 接下来，TGFbeta 的反义和显性失活将用于阻止野生型小鼠的纤维化。 这项工作是及时且令人兴奋的，因为它将利用我们新的小鼠肠内喂养模型，并使我们和其他人能够利用基因敲除和基因传递技术的力量来研究特定蛋白质和酶在酒精诱发的肝病中的作用。 这将使我们处于独特的地位，能够提供明确的新信息，填补我们在长期酒精性肝损伤机制方面的知识空白，并为临床试验奠定基础。