GENE THERAPY AND ALCOHOL-INDUCED FIBROSIS

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前兆。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TNFalpha参与早期酒精性肝损伤的明确证据。酒精性肝纤维化的机制尚不清楚，本提案的目的是利用我们用敲除小鼠和基因传递技术开发的新的肠内模型，在酒精性肝损伤的长期研究中填补我们知识的重要空白。我们将在这里使用基因敲除技术验证的统一假设是，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。首先，在Aim 1中，我们将通过确定野生型小鼠的病理包括纤维化来优化、表征和验证长期小鼠肠内酒精给药模型，野生型小鼠是选择用于研究的敲除的背景菌株。在Aim 2中，我们将利用我们的初步观察，即TNF受体1敲除既不产生脂肪也不产生炎症，来验证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敲除中的纤维化的影响，这些敲除将表现为脂肪但不炎症（ICAM-1敲除）和炎症但不脂肪（蛋白激酶A RIIbeta亚基敲除）。最后，在Aim 3中，我们将使用TGFbeta基因递送至TNF-R1敲除（即无脂肪或炎症），以验证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的反义和显性阴性将用于阻断野生型小鼠的纤维化。这项工作是及时和令人兴奋的，因为它将利用我们新的小鼠肠内喂养模型，并将允许我们和其他人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导的肝脏疾病中的作用。这将使我们独特地提供明确的新信息，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。

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.