Mechanisms of Intestinal Stem Cell Expansion Following Resection

描述（由申请人提供）：我们建立了一种小鼠回肠盲肠切除术（ICR）模型，并使用新的和传统的方法来定义介导适应性肠道生长的细胞机制，以补偿肠道损失。这些机制包括肠干细胞（ISC）在ICR后的短暂扩张，随后隐窝裂变和隐窝数量显著增加，从而介导粘膜表面积的增加。最近的报道表明，ISC可能有两个群体：位于Paneth细胞（Upper Stem cell Zone， USZ）上方的缓慢循环的静止ISC池（q-ISC）和以Lgr5表达为标志的隐窝基柱状细胞（CBC）的快速循环的活跃ISC池（a-ISC）。要完全了解适应性反应，需要分析这些假定的ISC亚群。我们的初步研究表明，在ICR后ISC扩张期间，肠道IGF-I表达增加。胰高血糖素样肽-2 （GLP-2）增加了观察到的ISC扩张和局部IGF-I表达，但仅在切除后立即给予。在临床环境中，许多患者在大量肠道丢失后无法耐受肠内营养（EN），需要全肠外营养（TPN）。慢性短肠综合征（SBS）在这些患者中的发病率增加表明tpn喂养可能会减弱正常的适应性ISC扩张。目前的提议检验了3种假设；1. 在ICR之后，q-ISC和a-ISC表现出不同的动力学，因为q-ISC的招募先于a-ISC的扩张。2. IGF-I的表达和信号传导是ICR诱导的正常扩张所必需的，并介导ICR后GLP-2的作用。构成型igf - 1的表达将增强和延长ICR后ISC的扩张。3. EN驱动IGF-I诱导ICR后的ISC扩增，但在tpn喂养的小鼠中，GLP-2会诱导局部IGF-I并恢复ICR后的ISC扩增。下面的情景分析将检验这些假设；SA1。利用Lgr5- lacz小鼠确定ICR是否诱导Lgr5/ lacz阳性a-ISC的扩增及其与隐窝裂变过程的关系。进一步的研究将通过定义连续胸苷类似物标记后USZ和CBC区域标签保留的时间过程来验证ICR后a-ISC和q-ISC的时间扩展。SA2。研究IGF-I组成性过表达或IGF-I信号通路受损的转基因小鼠ICR后ISC扩增和隐窝裂变。描述ICR + GLP-2后IGF-IR信号减弱小鼠ISC的扩增。SA3。随机给ICR小鼠提供en1 GLP-2与TPN 1 GLP-2的营养途径。评估营养途径和GLP-2给药对肠IGF-I表达和切除后正常ISC扩张的影响。公共卫生相关性：因损伤或疾病而手术切除的肠道可能导致无法吸收足够的液体和食物以维持生存。幸运的是，随着时间的推移，剩余的肠道通常能够弥补肠道的损失。当它不能，人们必须接受液体和营养静脉注射。本提案中概述的研究将帮助我们更好地理解每天不断更新肠道内膜的细胞（肠干细胞）如何增加数量以帮助弥补肠道损失。

DESCRIPTION (provided by applicant): We developed a mouse model of ileo-cecal resection (ICR) and used new and traditional approaches to define cellular mechanisms mediating adaptive intestinal growth to compensate for the intestinal loss. These mechanisms include a brief period of expansion of intestinal stem cells (ISC) immediately after ICR, followed by marked increases in crypt fission, and crypt number which mediate increases in mucosal surface area. Recent reports suggest that there may be two populations of ISC: a slowly cycling quiescent-ISC pool (q-ISC) located above Paneth cells (Upper Stem cell Zone, USZ) and a more rapidly cycling active-ISC pool (a-ISC) of crypt base columnar cells (CBC) marked by the expression of Lgr5. A complete understanding of the adaptive response will require analysis of these putative ISC sub-populations. Our preliminary studies demonstrate increased intestinal IGF-I expression during the period of ISC expansion after ICR. Glucagon-like peptide-2 (GLP-2) augmented the observed ISC expansion and local IGF-I expression, but only if given immediately following resection. In the clinical setting many patients are unable to tolerate enteral nutrition (EN) after massive intestinal loss, and require total parenteral nutrition (TPN). The increased incidence of chronic short bowel syndrome (SBS) in these patients suggests that TPN-feeding may attenuate the normal adaptive ISC expansion. The current proposal examines 3 hypotheses; 1. Following ICR, q- ISC and a-ISC show distinct kinetics, as recruitment of q-ISC will precede expansion of a-ISC. 2. IGF-I expression and signaling is required for normal ICR-induced expansion and mediates GLP-2 effects after ICR. Constitutive IGF-I expression will enhance and prolong ISC expansion following ICR. 3. EN drives IGF-I induced ISC expansion following ICR, but in TPN-fed mice GLP-2 will induce local IGF-I and restore ISC expansion following ICR. The following SA will test these hypotheses; SA1. Use the Lgr5-LacZ mouse to define whether ICR induces expansion of Lgr5/LacZ-positive a-ISC and how this relates to the process of crypt fission. Additional studies will validate the temporal expansion of a-ISC and q-ISC following ICR by defining the time course of label retention in the USZ and CBC regions after continuous thymidine analogue labeling. SA2. Study ISC expansion and crypt fission following ICR in transgenic mice with constitutive over-expression of IGF-I or impaired IGF-I signaling. Characterize expansion of ISC in mice with attenuated IGF-IR signaling following ICR + GLP-2. SA3. Randomize the route of providing nutrition to mice following ICR to EN 1 GLP-2 vs. TPN 1 GLP-2. Assess the influence of the route of nutrition and administration of GLP-2 on both intestinal expression of IGF-I and normal ISC expansion following resection. PUBLIC HEALTH RELEVANCE: Surgical loss of the intestine due to injury or disease may result in the inability to absorb enough liquid and food to survive. Fortunately, the remaining intestine is often able to compensate for this intestinal loss over time. When it cannot, people must receive both fluids and nutrition intravenously. Research outlined in this proposal will help us better understand how the cells that continually renew the lining of the intestine every day (intestinal stem cells) increase in number to help compensate following intestinal loss.