The Gastrointestinal Stem Cell Response to Injury

描述（由申请人提供）： 小肠上皮每3-5天更新一次。为了保持这种高的细胞损失率，新的细胞从多能肠干细胞（ISC）群体中不断产生。更全面地了解调控ISC功能的分子机制对于研究肠道损伤修复、正常稳态和胃肠道癌症具有巨大的意义。尽管ISC的确切身份和位置仍处于激烈的争论中，但最近使用优雅的报告小鼠模型的研究表明存在两种不同的ISC群体，一种是静止群体，位于潘氏细胞上方相对于隐窝底部的+4位置。另一种干细胞群体的特征在于散布在潘氏细胞之间的快速循环的隐窝基底柱状细胞（CBC）。这些+4细胞与辐射损伤（IR）后的“拯救”干细胞反应有关。我们以前曾报道过，推定的ISC标记Dclk 1（以前称为DCAMKL-1）主要表达在隐窝的+4位置，在基础条件下相对静止，并且与增殖干细胞（CBC）大部分不同。胃肠道干细胞越来越被认为是结肠癌的潜在起源细胞。已经证明，DCLK 1在人结直肠癌（CRC）中上调，并且可以用作预后标志物。有趣的是，siRNA介导的DCLK 1敲低导致结直肠肿瘤异种移植物生长停滞，通过let-7a miRNA抑制c-Myc、KRAS，通过miR-144抑制Notch-1，以及通过miR-200 a抑制上皮-间充质转化（EMT）相关因子。该VA Merit提案的中心假设是，Dclk 1在调节救援干细胞群以应对重度遗传毒性和潜在致突变性损伤中起关键作用，并且这些“救援/储备”细胞是可能导致胃肠道肿瘤的潜在起源细胞。为了验证这一假设，我们提出了3个具体的目标：目标1：确定Dclk 1是否通过Wnt和Notch信号转导，EMT和上皮屏障功能相关基因调节损伤后的小肠和结肠再生反应。Dclk 1基因敲除（Villin-Cre; Dclk 1flox/flox）和野生型小鼠将经受：（a）全身IR（遗传毒性损伤），以评估缺失+4拯救干细胞群在隐窝再生反应中的作用;（B）DSS诱导的结肠炎（细胞毒性损伤）和（c）氧化偶氮甲烷（AOM）+DSS（诱变性损伤）以评价上皮Dclk 1敲除在结肠隐窝再生和肿瘤发生中的作用。目标二：确定Dclk 1+细胞是否分别是遗传毒性/细胞毒性损伤和致突变损伤后再生隐窝的起源和癌症起源细胞。使用报告小鼠模型（Dclk 1-Cre-ERTM; Rosa 26 GFP），其中GFP在Dclk 1+细胞及其后代中表达，将进行谱系追踪研究。我们将研究遗传毒性，细胞毒性和致突变损伤对该小鼠模型的影响，以确定Dclk 1+细胞是否是再生隐窝和癌症的起源细胞。目的3：确定DCLK 1是否可以用作结直肠肿瘤（即腺瘤）检测的早期诊断标志物。在这里，我们将确定DCLK 1是否可以在结直肠肿瘤患者的循环中检测到，并与组织免疫染色相关。我们还将确定从人CRC血液样品中分离的DCLK 1+细胞的CSC性质。此外，DCLK 1可能用作早期结直肠肿瘤的组织和血清诊断标志物。CRC仍然是退伍军人人群发病率和死亡率的主要原因，并且有新的证据表明癌症干细胞是结肠癌的关键启动子。这些研究有很大的潜力来填补一个主要的未满足的医疗需求。

DESCRIPTION (provided by applicant): The small intestinal epithelium renews every 3-5 days. To maintain this high rate of cell loss, new cells are continuously generated from a population of multi-potent intestinal stem cells (ISCs). A more complete understanding of the molecular mechanisms that regulate ISC function has enormous implications for the study of gut injury repair, normal homeostasis and gastrointestinal cancer. Although the precise identity and location of ISCs remains under intense debate, recent studies using elegant reporter mouse models have suggested that two distinct ISC populations exist, one quiescent population, located at the +4 position relative to the crypt bottom just above the Paneth cells. The other stem cell population is characterized by rapidly cycling crypt base columnar cells (CBCs) interspersed between the Paneth cells. These +4 cells have been implicated in a "rescue" stem cell response following radiation injury (IR). We have previously reported that the putative ISC marker Dclk1 (formerly known as DCAMKL-1) is predominantly expressed in the +4 position in the crypt is relatively quiescent under basal conditions and is mostly distinct from the proliferative stem cells (CBCs). Gastrointestinal stem cells are increasingly being recognized as potential cells of origin in colon cancer. It has been demonstrated that DCLK1 is upregulated in human colorectal cancers (CRC) and can be used as a prognostic marker. Interestingly siRNA-mediated knockdown of DCLK1 results in colorectal tumor xenograft growth arrest, inhibition of c-Myc, KRAS via let-7a miRNA, Notch-1 via miR-144 and epithelial-mesenchymal transition (EMT) associated factors via miR-200a. The central hypothesis of this VA Merit proposal is that Dclk1 plays a key role in regulating the rescue stem cell population in response to severe genotoxic and potentially mutagenic injury, and these "rescue/reserve" cells are potential cells of origin that can contribute to gastrointestinal neoplasia. To test this hypothesis, we propose 3 specific aims: Aim 1: To determine whether Dclk1 regulates small intestinal and colonic regenerative responses after injury via genes related to Wnt and Notch signaling, EMT and epithelial barrier function. Dclk1 knockout (Villin-Cre; Dclk1flox/flox) and wild type mice will be subjected to: (a) whole body IR (genotoxic injury) to evaluate the effects of deletion of +4 rescue stem cell population in the crypt regenerative response; (b) DSS- induced colitis (cytotoxic injury) and (c) Azoxymethane (AOM)+DSS (mutagenic insult) to evaluate the effects of epithelial Dclk1 knockout in colonic crypt regeneration and tumorigenesis. Aim 2: To determine whether Dclk1+ cell is the origin of the regenerative crypt and cell of origin of cancer after genotoxic/cytotoxic injury and mutagenic insult respectively. Using a reporter mouse model (Dclk1-Cre-ERTM;Rosa26GFP), where GFP is expressed in Dclk1+ cells and their progeny, lineage tracing studies will be performed. We will examine the effects of genotoxic, cytotoxic and mutagenic injuries on this mouse model to determine whether the Dclk1+ cell is the cell of origin of regenerative crypts and cancer. Aim 3: To determine whether DCLK1 can be used as an early diagnostic marker for colorectal neoplasia (i.e. adenoma) detection. Here, we will determine whether DCLK1 can be detected in the circulation of patients with colorectal neoplasia and correlate with tissue immunostaining. We will also determine CSC properties of DCLK1+ cells isolated from human CRC blood samples. Furthermore, DCLK1 potentially can be used as a tissue and serum diagnostic marker of early colorectal neoplasia. CRC continues to be a major cause of morbidity and mortality in the Veteran population and there is emerging evidence that cancer stem cells are the key initiators of colon cancer. These studies have the major potential to fill a major unmet medical need.