The Gastrointestinal Stem Cell Response to Injury

小肠上皮每3-5天更新一次。为了保持如此高的细胞丢失率，新的细胞 从一群多能的肠道干细胞(ISCs)中连续产生。一个更完整的 了解调节ISC功能的分子机制对这项研究具有巨大的意义 肠道损伤修复、正常内环境稳定和胃肠道癌症。尽管准确的身份和位置 最近使用优雅的报告小鼠模型的研究表明，ISCs的作用仍处于激烈辩论中 存在两个不同的ISC群体，一个静止群体，位于相对于墓穴的+4位置 就在Paneth细胞上方的底部。另一种干细胞群体的特征是快速循环隐窝。 基柱状细胞(CBCs)散布在Paneth细胞之间。这些+4细胞与一种 辐射损伤(IR)后的“救援”干细胞反应。我们此前曾报道，推定的ISC 标记Dclk1(以前称为DCAMKL-1)主要在隐窝的+4位置表达 在基础条件下相对静止，与增殖干细胞(CBCs)大体不同。 胃肠道干细胞越来越被认为是结肠癌的潜在来源细胞。它有 已证实DCLK1在人类结直肠癌(CRC)中表达上调，并可作为一种 预后标记物。有趣的是，siRNA介导的DCLK1基因敲除导致结直肠肿瘤异种移植 生长停滞、抑制c-Myc、通过let-7a miRNA抑制KRAS、通过miR-144抑制Notch-1和抑制上皮间充质 通过miR-200A的过渡(EMT)相关因子。这项退伍军人福利计划的中心假设是 Dclk1在调节抢救干细胞数量以应对严重的遗传毒性方面起着关键作用 和潜在的突变损伤，而这些“抢救/储备”细胞是潜在的起源细胞，可以 会导致胃肠道肿瘤。为了验证这一假设，我们提出了三个具体目标：目标1： 确定Dclk1是否调节小肠和结肠的再生反应 通过与Wnt和Notch信号、EMT和上皮屏障功能相关的基因造成的损伤。Dclk1 (A)全身IR(基因毒性损伤)至 评估+4挽救干细胞群体缺失对隐窝再生反应的影响；(B)DSS- 诱导结肠炎(细胞毒性损伤)和(C)偶氮甲烷(AOM)+DSS(诱变伤害)以评估疗效 Dclk1基因敲除在结肠隐窝再生和肿瘤发生中的作用。目标2：确定是否 Dclk1+细胞是再生隐窝的起源，也是遗传毒性/细胞毒性后肿瘤的起源细胞 分别是伤害和诱变侮辱。使用报告小鼠模型(Dclk1-Cre-ERTM；Rosa26GFP)， 当GFP在Dclk1+细胞及其后代中表达时，将进行谱系追踪研究。我们会 检查遗传毒性、细胞毒性和诱变损伤对小鼠模型的影响，以确定 Dclk1+细胞是再生隐窝和癌症的起源细胞。目标3：确定DCLK1是否 可作为大肠肿瘤(即腺瘤)发现的早期诊断指标。在这里，我们 将确定是否可以在大肠肿瘤患者的循环中检测到DCLK1和 与组织免疫染色相关。我们还将确定从中国分离的DCLK1+细胞的CSC特性 人类CRC血样。此外，DCLK1还可用于组织和血清诊断 早期大肠肿瘤的标志物。儿童权利公约仍然是导致儿童发病率和死亡率的主要原因。 退伍军人群体，有新的证据表明，癌症干细胞是结肠的关键启动者 癌症。这些研究有很大的潜力来满足一个重大的未得到满足的医疗需求。

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.