Mechanisms of Intestinal Stem Cell Expansion Following Resection

描述(申请人提供)：我们开发了一个回肠盲肠切除术(ICR)的小鼠模型，并使用新的和传统的方法来定义调节适应性肠道生长的细胞机制，以补偿肠道的损失。这些机制包括ICR后肠干细胞(ISC)的短暂扩张，随后隐窝分裂显著增加，以及与粘膜表面积增加有关的隐窝数量。最近的报道表明，ISC可能有两个群体：位于Paneth细胞(上干细胞区，USZ)上方的慢周期静止ISC池(Q-ISC)和以Lgr5表达为标志的较快周期活动ISC池(a-ISC)的隐窝基底柱细胞(CBC)。要完全理解适应性反应，需要对这些假定的ISC亚群进行分析。我们的初步研究表明，ICR后ISC扩张期肠道IGF-I表达增加。胰升糖素样肽-2(GLP-2)增强了观察到的ISC扩张和局部IGF-I的表达，但仅在切除后立即给予。在临床环境下，许多患者在大量肠道失血后无法耐受肠内营养(EN)，需要完全肠外营养(TPN)。慢性短肠综合征(SBS)在这些患者中的发生率增加表明，TPN喂养可能会减弱正常的适应性ISC扩张。目前的建议检验了3个假说：1.在ICR之后，Q-ISC和a-ISC显示出不同的动力学，因为Q-ISC的招募将先于a-ISC的扩展。2.IGF-I的表达和信号转导是ICR诱导的正常扩张所必需的，并介导ICR后GLP-2的作用。ICR后结构性IGF-I的表达将促进和延长ISC的扩张。3.肠内营养促进ICR后IGF-I诱导的ISC扩张，而TPN喂养的小鼠GLP-2可诱导局部IGF-I并恢复ICR后ISC的扩张。下面的SA将检验这些假设；SA1。使用Lgr5-LacZ小鼠来定义ICR是否诱导Lgr5/LacZ阳性的a-ISC的扩张及其与隐窝分裂过程的关系。其他研究将通过定义连续胸苷类似物标记后USZ和CBC区域标记保留的时间进程来验证ICR后a-ISC和Q-ISC的时间扩展。SA2.研究IGF-I结构性过表达或IGF-I信号受损的转基因小鼠ICR后ISC扩张和隐窝分裂。ICR+GLP-2后IGF-IR信号减弱的小鼠ISC扩张特征。SA3.将ICR后向小鼠提供营养的途径随机分为EN 1 GLP-2和TPN 1 GLP-2。评估营养途径和给予GLP-2对切除后肠道IGF-I表达和正常ISC扩张的影响。公共卫生相关性：外科手术损伤或疾病造成的肠道丢失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这种肠道损失。如果不能，人们必须同时接受静脉注射的液体和营养。这项提案中概述的研究将帮助我们更好地了解每天不断更新肠道衬里的细胞(肠道干细胞)如何增加数量，以帮助弥补肠道丢失后的损失。

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.