Mechanisms of Gastrointestinal Epithelial Cell Injury & Repair During Development

由于发育不成熟，早产儿面临着许多独特的问题。最具破坏性的疾病之一是坏死性小肠结肠炎(NEC)，在美国，每年每10万名活产中就有12名婴儿死亡，幸存者经常出现严重的喂养问题、肝功能衰竭和神经发育障碍。了解发育过程中肠道损伤和修复的机制是开发NEC新的预防和治疗策略的关键。这项建议将通过检验早产儿和新生小鼠的肠道更容易受到肿瘤坏死因子诱导的损伤的假设，探讨发育过程中胃肠道上皮细胞损伤和修复的机制，这是因为个体发育正常的EGFR表达和激活减少所致。这一假说将通过以下具体目标来检验：1)确定肿瘤坏死因子在不同发育阶段对肠道损伤和细胞凋亡的影响。这将通过早期肠道损伤的组织病理损伤评分以及免疫荧光和免疫组织化学分析来完成；2)确定与成人相比，肿瘤坏死因子对新生儿EGFR抑制的影响。我们将研究肿瘤坏死因子对多个EGFR磷酸化位点和下游靶点的影响，以及EGFR内化在肿瘤坏死因子刺激的EGFR抑制中的作用。这一目标将利用免疫荧光和免疫印迹分析来研究EGFR激活的下游靶点；以及3)利用EGFR激活的药理学和遗传学模型来确定EGFR激活在保护肿瘤坏死因子诱导的损伤中的作用。这个目标将建立在目标1和目标2中开发的技术的基础上，并将它们应用于具有结构性活性的EGFR、药理活性的EGFR和缺乏EGFR的小鼠。总体而言，这些研究将揭示肿瘤坏死因子和表皮生长因子受体在肠道组织发育过程中的损伤过程中的作用，并将确定潜在的治疗和化学预防的新途径。除了上述研究，这项建议还将通过细胞生物学、发育生物学和统计学方面的教学培训，通过提供强大实验室的指导，以及通过利用Vanderbilt独特的强大资源来促进胃肠道疾病的独立研究，从而极大地促进职业发展。

Premature infants face a host of unique issues due to their developmental immaturity. One of the most devastating is necrotizing enterocolitis (NEC), which yearly kills 12 babies per 100,000 live births in the US and frequently leaves survivors with severe feeding issues, liver failure, and neurodevelopmental disability. Understanding mechanisms of intestinal injury and repair in developing intestine is key to developing new prevention and therapeutic strategies for NEC. This proposal will investigate the mechanisms of gastrointestinal epithelial cell injury and repair during development by testing the hypothesis that the intestine of premature infants and newborn mice is more susceptible to TNF-induced injury because of an ontogenically normal decrease in expression and activation of EGFR. This hypothesis will be examined through the following specific Aims: 1) Define the effects of TNF on intestinal injury and apoptosis at different developmental stages. This will be accomplished using histopathologic injury scores of early intestinal damage as well as immunofluorescence and immunohistochemical assays for apoptosis; 2) Determine the effects of TNF on EGFR inhibition in neonates compared to adults. We will study the effects of TNF on multiple EGFR phosphorylation sites and down-stream targets, and the role of EGFR internalization in TNF-stimulated EGFR inhibition. This aim will utilize immunofluorescence and western blot analysis to study the down-stream targets of EGFR activation; and 3) Determine the role of EGFR activation in protecting against TNF induced injury using pharmacologic and genetic models of EGFR activation. This aim will build on techniques developed in Aim 1 and 2 and apply them to mice with constitutively active EGFR, pharmacologically active EGFR, and deficiency of EGFR. Overall, these studies will reveal the roles of TNF and EGFR in intestinal injury processes in developing intestinal tissue, and will identify potential novel avenues of therapy and chemoprevention. In addition to the above studies, this proposal will greatly enhance career development through didactic training in cell biology, developmental biology, and statistics; by providing mentoring from a strong laboratory; and by utilizing the strong resources uniquely available at Vanderbilt to foster independent investigation in gastrointestinal disease.