Modeling oxidative stress and DNA damage using GI organotypic culture systems

描述（由申请人提供）：胃肠道的急性和慢性炎症环境，如胃食管反流病（GERD）、肠移植物抗宿主病（GVHD）、小肠移植后的急性排斥反应和IBD病症，如溃疡性结肠炎（UC），是相当常见的人类疾病。基于人类细胞和组织培养系统的疾病模型，其概括了体内生长和分化模式， 提高我们对疾病进展的认识，改进预防和检测策略。这是本提案的一个重要目标。 在这些疾病中，上皮细胞氧化应激是疾病进展的关键致病因素。 这种氧化应激是 部分来自内源性酶（环加氧酶和NADPH氧化酶），其诱导DNA损伤和突变，并改变DNA甲基化模式，其共同促成化生和癌症的发展。 我们已经开始探索这一点，使用器官型培养系统来模拟BE发病机制中的考克斯-2。 当我们异位表达考克斯-2在正常人食管角质形成细胞，我们观察到肠粘液填充囊肿的发展。 我们建议将这一成功扩展到生理学相关的方向。我们的主要目的是检验这一假设，即器官型培养系统可以被修改为模拟食管和肠上皮的急性和慢性氧化应激，其在生理上类似于GERD、BE、GVHD、小肠移植排斥和UC中的体内事件。 我们建议通过追求以下具体目标来测试这一点：1）调整食管器官型培养系统以更好地模拟GERD食管炎以及干细胞向化生和发育不良的进展。 2)开发器官型和3D多细胞培养系统，以模拟GVHD，小肠移植排斥反应和IBD（包括UC）的炎症微环境。 GERD/BE、IBD/肿瘤和GVHD是重要的、相对常见的疾病，给美国医疗保健系统带来了重大负担。我们建议开发新的多细胞体外人体组织工程模型，代表这些条件的发病机制。 这些模型将具有巨大的价值，使我们能够检验假设并快速推进我们对这些疾病的理解，并且由于考克斯-2的药理学抑制已经很好地建立，因此将产生转化影响。这项工作将大大提高我们研究，预防和治疗Barrett食管，IBD和GVHD的能力。 它还将促进开发新的治疗和预防策略，以改善这些重要临床疾病的患者护理。

DESCRIPTION (provided by applicant): Acute and chronic inflammatory environments of the gastrointestinal tract like gastroesophageal reflux disease (GERD), intestinal graft vs. host disease (GVHD), acute rejection after small intestine transplantation, and IBD conditions like Ulcerative colitis (UC), are fairly common human diseases. Disease models based on human cell and tissue culture systems that recapitulate in vivo growth and differentiation patterns would enhance our understanding of disease progression and improve prevention and detection strategies. This is an important objective of this proposal. In these disorders, epithelial cel oxidative stress is a key pathogenic factor for disease progression. This oxidative stress is partly from endogenous enzymes (Cyclooxygenases and NADPH oxidases) that induce DNA damage and mutations, and alter DNA methylation patterns, which together contributes to the development of metaplasia and cancer. We have begun to explore this using organotypic culture systems to model Cox-2 in BE pathogenesis. When we ectopically express Cox-2 in normal human esophageal keratinocytes, we observe the development of intestinal mucin-filled cysts. We propose to extend this success in physiologically relevant directions. Our main objective will be to test the hypothesis that the organotypic culture systems can be modified to model acute and chronic oxidative stress in esophageal and intestinal epithelium that physiologically resembles in vivo events in GERD, BE, GVHD, small bowel transplant rejection, and UC. We propose to test this by pursuing the following Specific Aims: 1) Adapt the esophageal organotypic culture system to better model GERD esophagitis and progression of stem cells to metaplasia and dysplasia. 2) Develop organotypic and 3D multi-cellular culture systems to model inflammatory microenvironments of GVHD, small bowel transplant rejection, and IBD including UC. GERD/BE, IBD/neoplasia, and GVHD are important, relatively common conditions that place a significant burden on the US healthcare system. We propose to develop novel multi-cellular in vitro human tissue engineered models that are representative of the pathogenesis for these conditions. These models will be of enormous value, allowing us to test hypotheses and advance our understanding of these disorders rapidly, and would have a translational impact since pharmacologic inhibition of Cox-2 is well established. This work will greatly improve our ability to study, prevent, and treat Barrett's esophagus, IBD, and GVHD. It will also foster the development of novel therapeutic and preventive strategies that will improve patient care for these important clinical conditions.