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间质细胞(ICC)存活、增殖和丢失的机制。正常的胃肠动力需要完整的ICC网络。ICC的缺失与几种胃肠动力障碍有关。尽管ICC在胃肠动力控制中起着重要的作用，但调控其存活、增殖和丢失的机制仍很不清楚。维持ICC数量需要在生存、增殖和丧失之间取得平衡。我们工作的主要主题是，5-羟色胺通过表达在ICC上的特定的5-HT受体，调节ICC存活、增殖和丢失之间的平衡。PI将通过使用ICC的原代培养和器官型培养、新鲜分离的人ICC和培养的小鼠ICC的膜片钳技术、同时记录ICC的机械活动和细胞内电活动的肌条、免疫组织化学、钙成像、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5-羟色胺在调节ICC存活、增殖和丢失中的作用，将检验三个假设：1)5-HT增加ICC的数量；2)ICC上表达5HT1、5HT2b、5HT3和5HT7受体；3)5-HT通过激活ICC上表达的调节细胞内钙处理的特定5HT受体来调节细胞的存活、增殖和丢失。这些假说得到了初步数据的支持，这些数据表明，5HT显著增加了细胞和器官培养中的ICC数量，ICC增殖，ICC表达死亡受体并经历细胞凋亡，这是调节组织稳态所必需的生理过程，人和小鼠ICC上表达特定的5HT受体，5HT调节ICC中的细胞内钙离子。建议研究的顺利完成具有基础意义和临床影响。这些研究结果将有助于更好地理解ICC数量调节的基本机制，同时也有助于更好地理解ICC丢失以及与ICC丢失相关的运动障碍的发生机制。事实上，基于我们的初步数据，早期的、定向的、基于5-羟色胺的治疗可能被认为是扭转运动障碍患者ICC丢失的一种机制。

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.