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先兆。重要的是，我们最近将Tsukamoto和French为大鼠开发的肠道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基因敲除，我们获得了明确的证据，证明TNFpha参与了早期酒精性肝损伤。酒精性肝纤维化的机制尚不清楚，本研究的目的是在酒精性肝损伤的长期研究中使用我们用基因敲除小鼠和基因输送技术开发的这种新的肠道模型，以填补我们知识中的重要空白。我们将在这里使用基因敲除技术测试的统一假设是，酒精诱导的纤维化需要炎症，而不是脂肪。我们认为这一作用是通过氧化剂实现的。首先，在目标1中，我们将通过确定包括纤维化在内的野生型小鼠的病理学来优化、表征和验证长期肠道酒精给药的小鼠模型，野生型小鼠是选定用于研究的基因敲除的背景菌株。在目标2中，我们将利用我们的初步观察，即肿瘤坏死因子受体1基因敲除既不发生脂肪也不发生炎症，以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脂肪而不是炎症(ICAM-1基因敲除)和炎症但不脂肪(蛋白激酶A RIIbeta亚基基因敲除)基因敲除的纤维化的影响。最后，在目标3中，我们将使用将转化生长因子β基因传递到肿瘤坏死因子-R1基因敲除(即没有脂肪或炎症)来检验酒精诱导的纤维化是由关键细胞因子转化生长因子β单独引起的假设。接下来，TGFbeta的反义和显性负性将被用来阻止野生型小鼠的纤维化。这项工作是及时和令人兴奋的，因为它将利用我们在小鼠身上的新肠道喂养模型，并将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导的肝病中的作用。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诱导的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.