Calcium Dynamics in Interstitial Cells of Cajal

描述（由申请人提供）：本提案的总体目标是确定控制Cajal （ICC）间质细胞存活、增殖和丧失的机制。正常的胃肠运动需要完整的ICC网络。ICC的丧失与几种胃肠运动障碍有关。尽管ICC在胃肠道运动的控制中发挥着重要作用，但调节其存活、增殖和丧失的机制仍在很大程度上是未知的。维持ICC数量需要在存活、增殖和损失之间取得平衡。我们工作的首要主题是5HT，通过在ICC上表达的特定5HT受体，调节ICC存活和增殖与ICC丧失之间的平衡。PI将通过使用ICC的原代培养和器官型培养，对新分离的人ICC和培养的小鼠ICC进行膜片钳技术，同时记录ICC机械活动和细胞内电活动的肌肉条，免疫组织化学，Ca2+成像，激光捕获显微解剖，电穿孔，Western blots, RT-PCR，单细胞PCR和定量PCR来验证这一中心假设。为了确定5HT对ICC存活、增殖和丧失的调控作用，我们将检验三个假设：1)5HT增加ICC的数量；2) 5HT1、5HT2B、5HT3、5HT7受体在ICC上表达；3) 5HT通过激活ICC上表达的特定5HT受体来调节细胞内Ca2+处理，从而调节细胞的存活、增殖和损失。这些假设得到了初步数据的支持，这些数据表明5HT显著增加细胞和器官型培养中的ICC数量，ICC增殖，ICC表达死亡受体并经历凋亡，这是调节组织稳态的必要生理过程，特异性5HT受体在人和小鼠ICC上表达，5HT调节ICC细胞内Ca2+。本研究的成功完成既有基础意义又有临床意义。这些研究的结果将有助于更好地了解调节ICC数量的基本机制，同时也有助于更好地了解导致ICC丧失和与ICC丧失相关的运动障碍发展的机制。事实上，根据我们的初步数据，早期的、定向的、基于5ht的治疗可能被提议作为一种机制来逆转运动障碍中ICC的丧失。

DESCRIPTION (provided by applicant): The overall objective of this proposal is to define the mechanisms that control survival, proliferation and loss of interstitial cells of Cajal (ICC). Normal gastrointestinal motility requires intact networks of ICC. Loss of ICC is associated with several disorders of gastrointestinal motility. Despite the prominent role ICC play in the control of gastrointestinal motility, the mechanisms that regulate their survival and proliferation and loss are still largely unknown. Maintenance of ICC numbers requires a balance between survival and proliferation and loss. The overarching theme of our work is that 5HT, through specific 5HT receptors expressed on ICC, regulates the balance between survival and proliferation of ICC and loss of ICC. The PI will test this central hypothesis by the use of primary cultures and organotypic cultures of ICC, patch clamp techniques on freshly dissociated human ICC and cultured mouse ICC, muscle strips to simultaneously record mechanical activity and intracellular electrical activity from ICC, immunohistochemistry, Ca2+ imaging, laser capture microdissection, electroporation, Western blots, RT-PCR, single cell PCR, and quantitative PCR. To determine the role of 5HT in the regulation of survival, proliferation and loss of ICC three hypotheses will be tested: 1) 5HT increases the number of ICC; 2) 5HT1, 5HT2B, 5HT3 and 5HT7 receptors are expressed on ICC; and 3) 5HT regulates cell survival, proliferation and loss by activation of specific 5HT receptors expressed on ICC that modulate intracellular Ca2+ handling. These hypotheses are supported by preliminary data that show that 5HT markedly increases ICC number in cellular and organotypic cultures, that ICC proliferate, that ICC express death receptors and undergo apoptosis, a necessary physiological process to regulate tissue homeostasis, that specific 5HT receptors are expressed on human and mouse ICC and that 5HT regulates intracellular Ca2+ in ICC. Successful completion of the proposed studies has both basic significance and clinical impact. The results of the studies will lead to a better understanding of the basic mechanisms that regulate the number of ICC while at the same time provide a better understanding of the mechanisms that contribute to loss of ICC and the development of motility disorders associated with ICC loss. Indeed, based on our preliminary data, early, directed, 5HT-based treatment may be proposed as a mechanism to reverse loss of ICC in motility disorders.