Calcium Dynamics in Interstitial Cells of Cajal

描述（由申请方提供）：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的丧失与几种胃肠动力障碍有关。尽管ICC在控制胃肠动力中发挥着重要作用，但调节其存活、增殖和丧失的机制在很大程度上仍不清楚。维持国际刑事法院的数目需要在生存、扩散和损失之间取得平衡。我们工作的首要主题是，5 HT，通过特定的5 HT受体表达的ICC，调节ICC的生存和增殖之间的平衡和ICC的损失。PI将通过使用ICC的原代培养物和器官型培养物、新鲜分离的人ICC和培养的小鼠ICC上的膜片钳技术、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、Ca 2+成像、激光捕获显微切割、电穿孔、蛋白质印迹、RT-PCR、单细胞PCR和定量PCR来检验这一中心假设。为了确定5 HT在ICC的存活、增殖和损失的调节中的作用，将测试三个假设：1）5 HT增加ICC的数量; 2）5 HT 1、5 HT 2B、5 HT 3和5 HT 7受体在ICC上表达;和3）5 HT通过激活在ICC上表达的调节细胞内Ca 2+处理的特异性5 HT受体来调节细胞存活、增殖和损失。这些假设支持的初步数据表明，5 HT显着增加ICC的细胞和器官型文化，ICC增殖，ICC表达死亡受体，并进行细胞凋亡，一个必要的生理过程，以调节组织的稳态，特定的5 HT受体表达的人类和小鼠ICC和5 HT调节细胞内Ca 2+在ICC。成功完成拟定研究具有基础意义和临床影响。这些研究的结果将导致更好地了解调控ICC数量的基本机制，同时更好地了解导致ICC丧失的机制以及与ICC丧失相关的运动障碍的发展。事实上，根据我们的初步数据，早期的，定向的，基于5 HT的治疗可能被提出作为一种机制，以扭转运动障碍的ICC损失。

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.