The role of Axin2+ stem cells in ulcer healing during colitis.

 描述(由申请人提供)： 患有炎症性肠病(IBD)的退伍军人接受结肠切除手术的一个主要原因是药物诱导的粘膜愈合失败。粘膜愈合是IBD治疗后恢复的一个可靠的临床标志，因为它与持久的临床缓解有关。尽管粘膜修复很重要，但我们对许多介导溃疡愈合的细胞和分子成分仍然知之甚少。目前的建议集中在结肠(上皮)干细胞(CSC)在结肠炎期间溃疡愈合中的作用。干细胞中的Wnt/β-Catenin信号促进细胞增殖和自我更新，这两种功能都是溃疡愈合和粘膜屏障重建所必需的。这一建议的一个主要支持者是假设Wnt/β-catenin信号在IBD的粘膜修复中起着不可或缺的作用，并且类固醇可以延迟溃疡愈合我受损的Wnt/β-catenin信号。在粘膜溃烂中，如IBD所见，表面粘膜被肉芽组织取代，肉芽组织再上皮化，形成新的隐窝结构。我们推测，从阶段1)产生肠上皮细胞(IEC)单层，到阶段2)形成上皮内陷，到阶段3)在溃疡表面形成隐窝岛，最后阶段4)成熟隐窝的再生，几个步骤都需要激活的Wnt信号。我们实验室的生化数据表明，Wnt/β-catenin信号在小鼠和IBD患者结肠炎溃疡愈合过程中增加。在询问Wnt信号在溃疡愈合中的作用的研究中，我们发现表达Wnt靶基因Axin2的IEC在溃疡边缘、溃疡表面和溃疡中部新形成的隐窝结构中扩张。使用一种新的Axin2报告鼠模型的研究表明，Axin2+IEC是多能性的，能够在体外从单个细胞生长出结肠样结构。总之，初步数据表明Axin2+IEC代表了一种新的ISC群体，该群体在溃疡愈合过程中形成了新的隐窝结构。在目标1中，Axin2lacZ/+和Axin2CreERT2/+；R26RmTmG/+小鼠将被用于研究溃疡相关Axin2+IEC的定位和这一新干细胞群体的基因表达谱，以及在DSS结肠炎溃疡愈合过程中进行谱系追踪。在目标2中，我们将检查使用类固醇治疗的小鼠的溃疡愈合情况。新的研究将利用Axin2-稳定的β-连环蛋白的特异性表达来研究它在类固醇治疗期间对溃疡愈合的作用。目的3项研究将探讨Axin2+IEC在人类IBD中的作用，并将分析类固醇影响溃疡愈合的机制。这些研究的目标将是确定未经治疗的UC患者在溃疡愈合区域是否表现出Axin2表达、Wnt信号和干细胞基因表达的增加(Aim 3A)，并在一项使用类固醇治疗结肠炎发作的UC患者的前瞻性试验中确定类固醇治疗是否降低了延迟溃疡愈合区域的Axin2表达和干细胞激活水平(Aim 3B)。总之，提出的研究将为控制结肠炎溃疡愈合的分子机制提供有价值的见解。目标是确定可以设计治疗靶点的区域，以安全地加速溃疡愈合并逆转类固醇对结肠炎粘膜修复的负面影响。

 DESCRIPTION (provided by applicant): A major cause of colon removal surgery in veterans with inflammatory bowel disease (IBD) is a failure of medically-induced mucosal healing. Mucosal healing is a reliable clinical marker of recovery after IBD therapy as it is associated with durable clinical remission. Despite the importance of mucosal repair, we continue to have a poor understanding of many of the cellular and molecular elements that mediate ulcer healing. The current proposal focuses on the role of colonic (epithelial) stem cells (CSC) in ulcer healing during colitis. Wnt/β-catenin signaling in stem cells promotes cell proliferation as well as self-renewal, both essential functions needed for ulcer healing and restitution of the mucosal barrier. A central tenant of this proposal is the hypothesis that Wnt/β-catenin signaling plays an integral role in mucosal repair in IBD and that steroids delay ulcer healing my impairing Wnt/β-catenin signaling. In mucosal ulceration, as seen in IBD, there is replacement of surface mucosa with granulation tissue that is re-epithelialized with new crypt structures. We suspect that active Wnt signaling is required for several steps through this sequence of events from stage 1) generation of an intestinal epithelial cell (IEC) monolayer, to stage 2) formation of epithelial invaginations, to stage 3) formation of crypt islands on ulcer surfaces and finally stage 4) regeneration of mature crypts (Fig 3). Biochemical data from our lab indicate Wnt/β-catenin signaling is increased during ulcer healing in colitis in mice and IBD patients. In studies to interrogate the role of Wnt signaling in ulcer healing, we discovered that IEC expressing mRNA for the Wnt target gene Axin2 expand in ulcer margins, on ulcer surfaces, and within newly-formed crypt structures in the middle of ulcers. Studies using a novel Axin2 reporter mouse model indicate that Axin2+ IEC are pluripotent and capable of growing colonoid structures in vitro from a single cell. Together the preliminary data suggest that Axin2+ IEC represent a novel ISC population that forms new crypt structures during ulcer healing. In Aim 1, Axin2lacZ/+ and Axin2 CreERT2/+; R26RmTmG/+ mice will be used to study ulcer-associated Axin2+ IEC localization and gene expression profiles of this new stem cell population, as well as perform lineage tracing during ulcer healing in DSS colitis. In Aim 2 we will examine ulcer healing in mice treated with steroids. New studies will utilize Axin2-specific expression of stabilized β-catenin to examine its role in ulcer healin during steroid therapy. Aim 3 studies will interrogate the role of Axin2+ IEC in human IBD and will analyze the mechanisms by which steroids impair ulcer healing. The goal of these studies will be to determine if untreated UC patients exhibit increased Axin2 expression, Wnt signaling and stem cell gene expression in areas of ulcer healing (Aim 3A) and determine whether steroid therapy reduces levels of Axin2 expression and stem cell activation in areas of delayed ulcer healing in a prospective trial of UC patients treated with steroids for a colitis flare (Aim 3B). Together the studies proposed will provide valuable insights into the molecular mechanisms that govern ulcer healing in colitis. The goal is to determine areas where therapeutic targets can be designed to safely accelerate ulcer healing and reverse the negative impact of steroids on mucosal repair in colitis.