MODULATION OF INTESTINAL HOMEOSTASIS AND CANCER

靶向调节正常分化和发育的关键发育途径 肠上皮细胞成熟是抑制肠易激综合征的根本途径 肠道肿瘤的发生。重要的原则证明已经出版，证明了 用分泌酶抑制剂抑制小鼠的Notch信号可以驱动肠道 癌细胞在体外和在ApcMin小鼠体内分化，后者 导致肿瘤停滞和消退。我们开发了一种新的小鼠遗传模型 它通过在小肠和大肠中特异性地针对Notch信号 消除使Notch受体岩藻糖基化的酶，这是 Notch与其配体的相互作用。这产生了一种戏剧性的表型 肠道：Noch信号下调，并有一个巨大的扩张 结肠和小肠中的杯状、盘状和内分泌分泌细胞谱系 4周龄时的肠壁，与增殖室扭曲平行。 这些小鼠存活下来，看起来很健康。然而，在9个月的时候，有一个非常大的 100%的小鼠肠黏膜的炎症反应，以及 发展为不典型增生和腺瘤形成。这些数据是第一次 Notch受体-配体特异性信号传递重要性的论证 肠粘膜动态平衡的相互作用。使用这个鼠标，我们的总体目标是 确定Notch信号在抑制肠道肿瘤中的作用 形成，这种抑制对肠道的长期后果是什么？ 动态平衡，使用营养的策略是否可以调节 缺口抑制，并了解Notch调节的机制 肠道内环境平衡及其与饮食信号的相互作用。 在目标1中，我们将把ApcMin等位基因引入这些小鼠以确定 Notch基因高效灭活抑制肿瘤形成的疗效观察 更长期的肿瘤抑制和绒毛引起的粘膜变化 肠上皮细胞中Notch的调节。此外，使用受监管的Villin-cre：er 转基因，我们将确定黏膜谱系特异性的动力学 分化和炎症反应在这些小鼠中发展，以及 肠道肿瘤随着时间的推移而复发，重复几轮Notch灭活的效果， 以及这对小鼠的粘膜和健康的长期影响。 描述了利用APC浮动鼠标和AOM模型的替代方案 结肠肿瘤的发生。在目标2中，我们将确定上皮细胞和 这些小鼠的炎症细胞反应在9个月内进化，特别是在 炎症程度，主要类炎性免疫细胞的表现 以及对细胞因子和其他生长因子的阐述；如何加快这一进程 西式饮食会增加肠道肿瘤形成的风险； 同时具有化学预防和抗炎作用的维生素D补充剂， 可以调节这些效应。最后，目标3将测试巨噬细胞如何从 对照小鼠，来自表现炎症的小鼠，以及来自炎症的小鼠 影响细胞生长的营养因素改变了反应，并 小鼠肠上皮细胞在体外和种植后的分化 活体肾胶囊。 这些实验将确定Notch下调的效果 抑制肠道肿瘤形成的信号转导机制及其长期效应 下调监管，对下调监管的反应是如何通过 影响人类结肠癌发展的重要营养因素，以及 肠道炎症的细胞和分子机制 同样受这些营养因素的影响。

Targeting of key developmental pathways that regulate the normal differentiation and maturation of intestinal epithelial cells is a fundamental approach for inhibition of intestinal tumorigenesis. Important proof-of-principle has been published demonstrating that inhibition of Notch signaling in mice with a ¿-secretase inhibitor can drive intestinal carcinoma cells to differentiate both in vitro and in the ApcMin mouse, in the latter case causing tumor stasis and regression. We have developed a new mouse genetic model that targets Notch signaling specifically in both the small and large intestine by eliminating the enzyme that fucosylates the Notch receptor, a step that is necessary for interaction of Notch with its ligands. This produces a dramatic phenotype in the intestine: Notch signaling is down-regulated, and there is a tremendous expansion of the goblet, Paneth and enteroendocrine secretory cell lineages in both the colon and small intestine at 4 weeks of age, paralleled by a distortion of the proliferative compartment. These mice survive and appear healthy. However, by 9 months there is a very large inflammatory response in the intestinal mucosa of 100% of the mice, and evidence for development of dysplasia and adenoma formation. These data are the first demonstration of the importance of signaling specifically through Notch receptor-ligand interaction for intestinal mucosal homeostasis. Using this mouse, our overall goals are to determine how effective Notch signaling is in inhibition of intestinal tumor formation, what the longer-term consequences of this inhibition are for intestinal homeostasis, whether strategies that employ nutrients can modulate effects of Notch inhibition, and to understand the mechanisms by which Notch regulates intestinal homeostasis and its interaction with signals from the diet. In Aim 1, we will we will introduce the ApcMin allele into these mice to determine the efficacy of inhibition of tumor formation by the efficient inactivation of Notch and the longer term tumor suppression and changes in the mucosa caused by the down- regulation of Notch in the intestinal epithelial cells. Further, using a regulated villin-cre:er transgene, we will determine the kinetics with which mucosal lineage specific differentiation and the inflammatory response develop in these mice, and whether intestinal tumors recur with time, the efficacy of repeated rounds of Notch inactivation, and the longer term influence of this on the mucosa and health of the mice. Alternatives are described that utilize an Apc floxed mouse and the AOM model of colon tumorigenesis. In Aim 2, we will determine how the epithelial cell and the inflammatory cell responses in these mice evolve over 9 months, especially in terms of extent of inflammation, representation of major classes of inflammatory immune cells and elaboration of cytokines and other growth factors; how this is accelerated and amplified by a western-style diet that elevates risk for intestinal tumor formation; and how vitamin D supplementation, which is both chemopreventive and anti-inflammatory, can modulate these effects. Finally, Aim 3 will assay how macrophages isolated from control mice, from mice exhibiting inflammation, and from mice in which the inflammatory response has been modified by nutritional factors influence cell growth and differentiation of mouse intestinal epithelial cells both in vitro and when implanted under the kidney capsule in vivo. These experiments will determine the efficacy of down-regulation of Notch signaling in inhibiting intestinal tumor formation and the longer term effects of such down-regulation, how the responses to Notch down regulation are modulated by important nutritional factors that influence development of human colon cancer, and cellular and molecular mechanisms by which the inflammation of the intestine can be modulated by these same nutritional factors.