Hypoxia and free radicals in alcoholic pancreatitis

申请人摘要： 我们假设乙醇诱导的慢性胰腺炎是由 缺氧/复氧损伤，导致炎症细胞刺激 循环内毒素对这些细胞的募集和激活。我们 近年来表现出慢性胰腺炎胰腺损伤的特点 在肠道模型中，人类被修改为提供更高数量的乙醇。我们的 第一个目标是描述这种新模式的特点。Wistar大鼠将接受酒精 在内部使用我们修改后的方案长达6个月。胰腺功能 损伤将被确定，并与关键分子事件的变化进行比较。 我们预计酒精将导致渐进性的、不可逆转的变化 胰腺形态和功能，与临床观察相似。我们会 接下来，测试酒精会导致活体胰腺缺氧的假设。 最初，我们将确定缺氧标志物匹莫硝唑是否可以用于 大鼠胰腺缺氧指数。然后我们将确定酒精的影响 在我们的肠内模型中胰腺缺氧。我们预计慢性乙醇 会导致早期缺氧的增加，这表明缺氧可能对 在造成伤害方面起到关键的早期作用。清除内毒素的效果观察 通过乳酸菌治疗取代革兰氏阴性菌，或杀死 接下来将研究含有二氧化硅的巨噬细胞。我们预计胰腺损伤将 被这些治疗钝化，表明内毒素和巨噬细胞的作用 慢性酒精性胰腺炎。接下来，自由基假说 在慢性酒精性胰腺炎中起到致病作用将得到检验。这 问题将使用腺相关病毒(RAAV)来解决 超氧化物歧化酶(SOD)和导致稳定的长期胰腺表达。 首先，允许胰腺转基因表达的最佳条件是 下定决心。含超氧化物歧化酶的重组腺相关病毒对血管内皮细胞生长的影响 胰腺损伤将被确定。自由基的形成与氧化 压力也将被量化。我们预计，防止氧化应激 使用超氧化物歧化酶可以预防慢性酒精性胰腺炎，这表明 自由基在慢性酒精中毒进展中的重要作用 胰腺炎。这项工作将为今后的研究奠定力学基础 预防酒精性胰腺炎的靶向疗法可应用于 诊所，那里迫切需要这样的治疗。此外，通过 授课培训以及与他的导师和主要教员的互动 酒精暴露的肠道模型、分子生物学和病毒载体基因 治疗，申请者将获得新的技能，这些技能将极大地提高他的 职业发展，并弥合差距，成为成功的成员 学术界和科学界。

APPLICANT'S ABSTRACT: We hypothesize that ethanol-induced chronic pancreatitis is triggered by hypoxia/reoxygenation injury, leading to stimulation of inflammatory cell recruitment and activation of these cells by circulating endotoxins. We recently showed pancreatic injury characteristic of chronic pancreatitis in humans in an enteral model modified to deliver higher amounts of ethanol. Our first goal is to characterize this new model. Wistar rats will receive alcohol internally using our modified protocol for up to 6 months. Pancreatic function and injury will be determined and compared to changes in key molecular events. We expect that alcohol will cause progressive, irreversible changes in pancreatic morphology and function, similar to clinical observations. We will next test the hypothesis that alcohol causes hypoxia in pancreas in vivo. Initially, we will determine if the hypoxia marker pimonidazole can be used to index pancreatic hypoxia in rats. We will then determine the effect of alcohol on pancreatic hypoxia in our enteral model. We expect that chronic ethanol will cause early increases in hypoxia, suggesting that hypoxia might play a key early role in the initiation of damage. The effect of removing endotoxin by lactobacillus treatment to displace Gram-negative bacteria, or killing macrophages with silica will next be studied. We expect pancreatic damage will be blunted by these treatments, suggesting a role of endotoxin and macrophages in chronic alcoholic pancreatitis. Next, the hypothesis that free radicals play a causative role in chronic alcoholic pancreatitis will be tested. This question will be addressed using adeno-associated virus (RAAV) to deliver superoxide dismutase (SOD) and cause stable long-term pancreatic expression. First, optimal conditions to confer pancreatic transgene expression will be determined. The effect of RAAV containing superoxide dismutase (SOD) on pancreatic injury will be determined. Free radical formation and oxidative stress will also be quantitated. We expect that preventing oxidative stress with SOD will protect against chronic alcoholic pancreatitis, indicating that free radicals play an important role in the progression of chronic alcoholic pancreatitis. This work will lay the mechanistic foundation for future studies of targeted therapies to prevent alcoholic pancreatitis that can applied in the clinic, where such therapy is desperately needed. Further, through didactic training and interactions with his mentor and key faculty in the enteral model of alcohol exposure, molecular biology, and viral vector gene therapy, the applicant will acquire new skills that will greatly enhance his career development and bridge the gap to becoming a successful member of the academic and scientific community.