Functional role of fibroblast-like cells in GI muscles

描述（由申请人提供）：胃肠道（GI）肌肉中的神经肌肉装置比以前理解的更复杂。肠运动神经末梢与平滑肌细胞（SMC）、Cajal间质细胞（ICC）和成纤维细胞样细胞（FLC）有密切接触。ICC和FLC都电耦合到SMC。因此，ICC和FLC转导的信号可以通过SMC合胞体电传递。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。本项目将评价FLC在小鼠和人胃肠道肌肉中的功能。FLC用pdgfr - α抗体特异性标记。我们已经开发并验证了在pdgfr - α +细胞中具有eGFP组成表达的转基因小鼠模型。我们将分离pdgfr - α +细胞，利用膜片钳技术研究这些细胞表达的内源性电导，并表征肠内神经递质对膜电导的影响。初步研究表明，嘌呤类神经递质激活了一个大的外向电流，并将对该电流的特性和药理学进行研究。SMC和ICC响应嘌呤递质与激活净内向电流。因此，我们的数据表明pdgfr - α细胞介导胃肠道肌肉的嘌呤能神经反应。我们将利用Ca2+染料的荧光研究PDGFR +细胞的神经支配。我们还将使用荧光激活细胞分选纯化PDGFR- α +细胞并表征PDGFR +细胞的转录组。丢失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的神经调节的理解，并可能为运动和功能性GI的治疗提供新的靶点

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.