Interstitial cells of Cajal in diabetic gastropathy

描述（申请人提供）：存在于许多器官中的体细胞干细胞负责组织的维持和修复，也有助于衰老和癌症。虽然干细胞和细胞更新在快速更新组织中的作用是明显的，但它们在胃肠道神经肌肉室中的重要性直到最近才被认识到。包括糖尿病性胃轻瘫在内的胃肠运动障碍的经典观点是，它们是由于组织功能受损和终末分化细胞类型（如肠神经元、平滑肌细胞和Cajal间质细胞（ICC））的变性所致。这也反映在目前的治疗方式上，其重点是症状控制和刺激残余功能。然而，这些治疗并不能治愈，而且在很大程度上是无效的。最近的研究表明，出生后肠神经元、平滑肌细胞和ICC具有相当大的可塑性，这表明这种旧观点是站不住脚的。我们最近在成年小鼠的胃肌肉组织中发现了一种可以产生ICC （ICC- sc）的干细胞。这种模式的转变为揭示ICC维持和分化的机制以及开发新的、合理的疗法来再生这些疾病中的内源性或移植组织干细胞的ICC网络开辟了全新的机会。然而，在实现这一目标之前，必须克服几个障碍，包括缺乏对控制ICC-SC自我更新和分化的机制的理解，以及组织微环境在这些过程中的作用。因此，当前项目的总体目标是确定控制ICC-SC增殖和分化的关键细胞内在和细胞外在因素，并在临床前疾病模型中通过这些因素测试靶向ICC-SC的药理学药物。我们的第一个具体目标是确定多梳蛋白作为细胞内在因子在ICC-SC维持和分化的表观遗传控制中的作用和作用机制，并测试间接组蛋白甲基转移酶抑制剂在体内的效用，通过抑制多梳活性，刺激移植和内源性ICC-SC的分化。我们的第二个具体目标是确定糖原合成酶激酶3 （Gsk3）作为通过平滑肌细胞干细胞因子表达调节ICC-SC的外部因子的作用和分子作用机制，并确定Gsk3抑制剂通过支持ICC-SC自我更新和分化来逆转胃轻瘫的体内实用性。我们将使用我们实验室开发的新模型和新技术，包括新鲜分离和培养的ICC- sc，遗传标记的ICC- sc移植，以及流式细胞术对ICC发展的关键阶段进行定量分析。我们还将采用最先进的分子生物学和重组DNA技术来研究基因转录的表观遗传控制和用于临床前测试的小鼠模型。拟议的研究将确定运动性疾病中ICC丧失的新药理学干预的机制基础，并为临床试验奠定基础。

DESCRIPTION (provided by applicant): Somatic stem cells residing in many organs are responsible for tissue maintenance and repair and also contribute to aging and cancer. While the role of stem cells and cell turnover is obvious in rapidly renewing tissues, their significance in the gastrointestinal neuromuscular compartment has only recently been recognized. The classic view of gastrointestinal motility disorders including diabetic gastroparesis is that they are due to impaired tissue function and degeneration of terminally differentiated cell types such as enteric neurons, smooth muscle cells and interstitial cells of Cajal (ICC). This is also reflected in current treatment modalities, which focus on symptom control and stimulation of residual function. However, these treatments are not curative and to a large extent ineffective. Recent studies demonstrating considerable plasticity of postnatal enteric neurons, smooth muscle cells and ICC indicate that this old view is untenable. We recently identified a stem cell residing in the gastric musculature of adult mice that can give rise to ICC (ICC-SC). This paradigm shift has opened entirely new opportunities to unravel the mechanisms of ICC maintenance and differentiation and to develop new, rational therapies to regenerate ICC networks from endogenous or transplanted tissue stem cells in these disorders. However, there are several barriers that must be overcome before this goal can be realized including a lack of understanding of the mechanisms that control ICC-SC self- renewal and differentiation, as well as the role of the tissue microenvironment in these processes. Therefore, the overall goals of the current project are to determine the key cell-intrinsic and cell-extrinsic factors that control ICC-SC proliferation and differentiation and to test pharmacological agents that target ICC-SC via these factors in preclinical disease models. Our first specific aim is to determine the role and mechanisms-of-action of polycomb group proteins as cell-intrinsic factors in the epigenetic control of ICC-SC maintenance and differentiation and to test the in vivo utility of indirect histone methyltransferase inhibitors to stimulate, through the inhibition of polycomb activity, the differentiation of transplanted and endogenous ICC-SC. Our second specific aim is to determine the role and molecular mechanisms-of-action of glycogen synthase kinase 3 (Gsk3) as an extrinsic factor in regulating ICC-SC through stem cell factor expression in smooth muscle cells and to determine the in vivo utility of Gsk3 inhibitors to reverse gastroparesis by supporting ICC-SC self- renewal and differentiation. We will use novel models and new technology developed in our laboratory including freshly isolated and cultured ICC-SC, transplantation of genetically labeled ICC-SC, and quantitative analysis of key stages of ICC development by flow cytometry. We will also employ state-of-the-art molecular biological and recombinant DNA techniques to study epigenetic control of gene transcription and mouse models for preclinical testing. The proposed studies will determine the mechanistic basis for, and set the stage for clinical trials of, novel pharmacological interventions for loss of ICC in motility diseases.