PEROXYNITRITE IN ENTEROCYTE APOPTOSIS & CELL SIGNALLING

肠道屏障功能障碍和细菌易位是导致危重病患者医院感染的重要因素。福特博士实验室以前的工作表明，在内毒素血症动物模型和人类坏死性小肠结肠炎模型中，诱导型一氧化氮合酶(INOS)的持续上调、肠细胞的凋亡以及对过氧亚硝酸根(PN)的3-硝基酪氨酸的免疫反应共同位于肠道粘膜中。这表明，在这些炎症条件下，iNOS持续过表达，通过一氧化氮(NO)和PN的形成导致肠细胞凋亡。除了诱导细胞凋亡外，PN还可以抑制依赖于酪氨酸磷酸化的肠细胞增殖信号通路，如酪氨酸激酶的Src家族。PN能够硝化蛋白质中的酪氨酸残留物，从而阻止酪氨酸的磷酸化。最近的一些报道表明，NO和PN能够阻止细胞信号通路中涉及的各种蛋白质的酪氨酸磷酸化。因此，假设PN导致肠细胞凋亡增加和肠细胞增殖减少或静止之间的失衡，从而导致粘膜剥脱和细菌移位。本研究的第一个目的是在体外研究肠外营养诱导肠上皮细胞凋亡的分子机制。第二个目标是研究肠细胞信号通路，特别是Src酪氨酸激酶通路和通过表皮生长因子受体的信号，并在分子水平上确定这些通路是如何受到PN的影响的。

Gut barrier failure and bacterial translocation are important factors leading to nosocomial infections in critically ill patients. Previous work from Dr. Ford's lab has shown that sustained upregulation of inducible nitric oxide synthase (iNOS), enterocyte apoptosis, and immunoreactivity to 3-nitrotyrosine, afoot-print' of peroxynitrite (PN), co-localize in the intestinal mucosa in animal models of endotoxemia and in human necrotizing enterocolitis. This suggests that the sustained over-expression of iNOS occurring in these inflammatory conditions leads to enterocyte apoptosis through nitric oxide (NO) and PN formation. In addition to inducing apoptosis, PN may also inhibit enterocyte proliferative cell signaling pathways that are dependent on tyrosine phosphorylation, such as the Src family of tyrosine kinases. PN is able to nitrate tyrosine residues in proteins and thus prevent tyrosine phosphorylation. Several recent reports have shown that NO and PN are able to prevent tyrosine phosphorylation of various proteins involved in cell signaling pathways. It is hypothesized, therefore, that PN causes an imbalance between increased enterocyte apoptosis and decreased or static enterocyte proliferation which leads to mucosal denudation and bacterial translocation. The first goal of this proposal is to study the molecular mechanisms of PN-induced enterocyte apoptosis in vitro. The second goal is to study enterocyte cell signaling pathways, in particular Src tyrosine kinase pathways and signaling via the epidermal growth factor receptor, and to determine how these pathways are affected on a molecular level by PN.