MODULATION OF INTESTINAL HOMEOSTASIS AND CANCER

靶向调节正常分化的关键发育途径， 肠上皮细胞的成熟是抑制肠上皮细胞增殖的基本途径。 肠道肿瘤发生已经发表了重要的原理证明， 用分泌酶抑制剂抑制小鼠的Notch信号， 癌细胞在体外和ApcMin小鼠中分化，在后者的情况下 导致肿瘤停滞和退化。我们开发了一种新的小鼠遗传模型 在小肠和大肠中特异性靶向Notch信号传导， 消除岩藻糖基化Notch受体的酶，这是一个必要的步骤， Notch与其配体的相互作用。这会产生一个戏剧性的表型， 肠：Notch信号转导下调，并且有一个巨大的扩张， 在结肠和小肠中， 肠在4周龄时，由增生室的扭曲所包围。 这些老鼠存活下来，看起来很健康。然而，到9个月时， 100%小鼠肠粘膜中的炎症反应，以及 发育异常和腺瘤形成。这些数据是第一个 证明通过Notch受体-配体特异性信号传导的重要性 肠粘膜稳态的相互作用。使用这款鼠标，我们的总体目标是 为了确定Notch信号传导在抑制肠肿瘤中的有效性， 形成，这种抑制的长期后果是什么肠道 稳态，是否策略，采用营养素可以调节的影响， Notch抑制，并了解Notch调节的机制 肠道内稳态及其与饮食信号的相互作用。 在目标1中，我们将ApcMin等位基因引入这些小鼠中以确定 通过Notch的有效失活抑制肿瘤形成的功效以及通过Notch的有效失活抑制肿瘤形成的功效。 长期的肿瘤抑制和粘膜的变化引起的下降， 肠上皮细胞中Notch的调节。此外，使用调节的villin-cre：er 转基因，我们将确定与粘膜谱系特异性 分化和炎症反应在这些小鼠中发展，以及 肠肿瘤随时间复发，重复轮的Notch失活的功效， 以及这对小鼠的粘膜和健康的长期影响。 描述了利用Apc floxed小鼠和A0 M模型的替代方案。 结肠肿瘤发生在目标2中，我们将确定上皮细胞和上皮细胞是如何被激活的。 这些小鼠的炎症细胞反应在9个月内逐渐发展，特别是在 炎症程度，炎症免疫细胞主要类别的代表 以及细胞因子和其他生长因子的加工;这是如何加速的， 西式饮食会增加肠道肿瘤形成的风险; 补充维生素D，既有化学预防作用又有抗炎作用， 可以调节这些效应。最后，Aim 3将分析巨噬细胞如何从 对照小鼠，来自表现出炎症的小鼠，以及来自其中炎性细胞因子被抑制的小鼠。 营养因素影响细胞生长， 小鼠肠上皮细胞在体外和植入时的分化 体内的肾囊。 这些实验将确定下调Notch的功效， 在抑制肠肿瘤形成中的信号传导和这种信号传导的长期作用 下调，对Notch下调的反应如何通过 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， 由这些相同的营养因素调节。

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.