Functional role of fibroblast-like cells in GI muscles

描述(申请人提供)：胃肠道(GI)肌肉中的神经肌肉装置比以前所了解的要复杂得多。肠运动神经末梢与3种细胞有非常密切的接触：平滑肌细胞(SMC)、Cajal间质细胞(ICC)和成纤维细胞样细胞(FLC)。ICC和FLC都与SMC电耦合。因此，由ICC和FLC转导的信号可以通过SMC的合胞体进行电传递。以往对SMC和ICC的研究较多，但对FLC在胃肠道器官中的功能研究较少。本项目将评估FLC在小鼠和人胃肠道肌肉中的功能。FLC被特异性地标记有PDGFR-α抗体。我们已经建立并验证了一种在PDGFR-α+细胞中结构性表达EGFP的转基因小鼠模型。我们将分离PDGFR-α+细胞，并使用膜片钳技术来研究这些细胞表达的内源性电导，并表征肠神经递质对膜电导的影响。初步研究表明，嘌呤神经递质激活了一个大的外向电流，这一电流的特征和药理学将被研究。SMC和ICC通过激活净内向电流对嘌呤递质作出反应。因此，我们的数据表明PDGFR-α细胞在胃肠道肌肉中介导嘌呤能神经反应。我们将利用钙离子染料的荧光来研究PDGFR+细胞的神经支配。我们还将使用荧光激活的细胞分选来纯化PDGFR-α+细胞，并对PDGFR+细胞的转录组进行鉴定。失去PDGFR+细胞对嘌呤能反应的影响也将被研究。这些研究将提高我们对结肠运动神经调节的理解，并可能为运动和功能性胃肠道疾病的治疗提供新的靶点。 精神错乱。

DESCRIPTION (provided by applicant): The neuromuscular apparatus in gastrointestinal (GI) muscles is more complicated than previously understood. Enteric motor nerve terminals make very close contacts with 3 types of cells: smooth muscle cells (SMC), interstitial cells of Cajal (ICC) and flibroblast-like cells (FLC). Both ICC and FLC are electrically coupled to SMC. Thus, signals transduced by ICC and FLC can be conveyed electrically throughout the syncytium of SMC. Considerable study has been directed toward SMC and ICC in the past, but there are few studies that have investigated the function of FLC in GI organs. This project will evaluate the function of FLC in mouse and human GI muscles. FLC are specifically labeled with antibodies for PDGFR-alpha. We have developed and verified a transgenic mouse model with constitutive expression of eGFP in PDGFR-alpha+ cells. We will isolate the PDGFR-alpha+ cells and use patch clamp techniques to study the endogenous conductances expressed by these cells and characterize the effects of enteric neurotransmitters on membrane conductances. Preliminary studies demonstrate a large outward current activated by purine neurotransmitters, and the characteristics and pharmacology of this current will be investigated. SMC and ICC respond to purine transmitters with activation of net inward current. Thus, our data suggest that PDGFR-alpha cells mediate purinergic neural responses in GI muscles. We will investigate the innervation of PDGFR + cells using the fluorescence of Ca2+ dyes. We will also use fluorescence activated cell sorting to purify PDGFR-alpha+ cells and characterize the transcriptome of PDGFR + cells. The effects of losing PDGFR + cells on purinergic responses will also be investigated. These studies will improve our understanding of neural regulation of colonic motility and will possibly provide new targets for therapies for motility and functional GI disorders.