Mechanisms of Intestinal Stem Cell Expansion Following Resection

描述（由申请人提供）：我们开发了回盲肠切除术（ICR）的小鼠模型，并使用新的和传统的方法来定义介导适应性肠道生长以补偿肠道损失的细胞机制。这些机制包括ICR后肠干细胞（ISC）的短暂扩增，随后隐窝分裂和隐窝数量显著增加，介导粘膜表面积增加。最近的报道表明，可能存在两个群体的ISC：位于潘氏细胞（上干细胞区，USZ）上方的缓慢循环的静止ISC池（q-ISC）和由Lgr 5表达标记的隐窝基底柱状细胞（CBC）的更快速循环的活性ISC池（a-ISC）。一个完整的理解的适应性反应将需要分析这些推定的ISC亚群。我们的初步研究表明，增加肠道IGF-I的表达在期间的ISC扩张后ICR。胰高血糖素样肽-2（GLP-2）增强了观察到的ISC扩张和局部IGF-I表达，但仅在切除后立即给予。在临床上，许多患者在大量肠道丢失后不能耐受肠内营养（EN），而需要全肠外营养（TPN）。这些患者中慢性短肠综合征（SBS）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。目前的建议审查3个假设; 1。在ICR之后，q-ISC和a-ISC显示出不同的动力学，因为q-ISC的募集将先于a-ISC的扩增。2. IGF-I表达和信号传导是正常ICR诱导的扩增所必需的，并在ICR后介导GLP-2效应。组成性IGF-I表达将增强并延长ICR后ISC的扩张。3. EN驱动ICR后IGF-I诱导的ISC扩增，但在TPN喂养的小鼠中，GLP-2将诱导局部IGF-I并恢复ICR后的ISC扩增。下面的SA将测试这些假设; SA 1。使用Lgr 5-LacZ小鼠确定ICR是否诱导Lgr 5/LacZ阳性a-ISC的扩增以及这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区域中标记保留的时间过程来验证ICR后a-ISC和q-ISC的时间扩展。SA 2.在IGF-I组成性过表达或IGF-I信号传导受损的转基因小鼠中研究ICR后ISC扩增和隐窝分裂。表征ICR + GLP-2后IGF-1 R信号转导减弱的小鼠中ISC的扩增。SA 3.随机分配ICR后EN 1 GLP-2与TPN 1 GLP-2的小鼠营养供应途径。评估营养途径和GLP-2给药对切除术后肠IGF-I表达和正常ISC扩张的影响。公共卫生相关性：由于受伤或疾病导致的肠道手术损失可能导致无法吸收足够的液体和食物来生存。幸运的是，随着时间的推移，剩余的肠道通常能够补偿这种肠道损失。如果不能，人们必须通过静脉注射接受液体和营养。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内壁的细胞（肠道干细胞）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.