Mechanisms of Gastrointestinal Epithelial Cell Injury & Repair During Development

早产儿由于发育不成熟而面临许多独特的问题。其中最具破坏性的是坏死性小肠结肠炎（NEC），在美国，每年每10万名活产婴儿中就有12名死亡，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。了解发育中的肠道损伤和修复机制是开发新的NEC预防和治疗策略的关键。本研究将研究发育过程中胃肠道上皮细胞损伤和修复的机制，通过验证一个假设，即早产儿和新生小鼠的肠道更容易受到tnf诱导的损伤，因为EGFR的表达和激活在个体上正常下降。我们将通过以下具体目的来检验这一假设：1)明确TNF对不同发育阶段肠道损伤和细胞凋亡的影响。这将通过早期肠损伤的组织病理学损伤评分以及细胞凋亡的免疫荧光和免疫组织化学分析来完成；2)比较TNF对新生儿和成人EGFR抑制的影响。我们将研究TNF对多个EGFR磷酸化位点和下游靶点的影响，以及EGFR内化在TNF刺激的EGFR抑制中的作用。本研究将利用免疫荧光和western blot分析研究EGFR激活的下游靶点；3)利用EGFR激活的药理学和遗传学模型确定EGFR激活在保护TNF诱导的损伤中的作用。该目标将建立在目标1和目标2中开发的技术基础上，并将其应用于具有组成性活性EGFR、药理活性EGFR和缺乏EGFR的小鼠。总的来说，这些研究将揭示TNF和EGFR在肠组织发育中肠损伤过程中的作用，并将确定治疗和化学预防的潜在新途径。除上述研究外，本计划将透过细胞生物学、发育生物学及统计学的教学训练，大大促进职业发展；通过提供来自强大实验室的指导；并利用范德比尔特大学独特的强大资源来促进胃肠疾病的独立研究。

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.