STIM 1/Orai 1 信号通路在高血压负荷诱导冠状动脉血管平滑肌细胞异常增殖及迁移的机制研究

It is well appreciated that hypertension contributes to coronary atherosclerosis by inducing abnormal proliferation and migration of vascular smooth muscle cells (VSMC) through disordering intracellular calcium regulation. STIM 1/Orai 1 is an important channel of calcium influx in VSMC. Based on previous publications and our preliminary data, we put the hypothesis that sustained hypertensive state results in the disorder of calcium regulation in coronary arterial smooth muscle cells (CASMC) by increasing expression of STIM 1/ Orai 1 via activation of calcineurin-NFAT signal. On the premise of clarifying the difference of STIM 1/ Orai 1 expression and NFAT nuclear accumulation in CASMC between wistar-kyoto (WKY) and spontaneously hypertensive rats, we propose to apply high hydrostatic pressure cell model as hypertensive state in vitro. Series experiments, such as co-immunoprecipitation , confocal microscopy, path clamp, Transwell will be utilized to illustrate the effect of interactions between STIM 1and Orai 1 on calcium influx, and the proliferation and migration of CASMC under hypertensive state. Furthermore, signaling transduction pathway inhibitors and gene-silencing technology will be applied to verify the regulation of calcineurin-NFAT signal on STIM 1/ Orai 1. In short, this project aims to clarify the effect and mechanism of hypertension on coronary atherosclerosis, and will provide theoretical basis for a novel therapeutic strategy in the clinical treatment of coronary artery disease.

高血压促使胞内钙调控紊乱诱导血管平滑肌细胞异常增殖迁移是冠状动脉粥样硬化发生发展的关键环节。STIM1/Orai1是血管平滑肌细胞钙内流的重要通道。在总结前人和自身前期基础上我们提出假设持续高血压状态通过激活calcineurin-NFAT信号上调STIM 1/Orai 1表达致冠状动脉平滑肌细胞（CASMC）钙调控紊乱。在明确WKY及自发性高血压大鼠CASMC中STIM1/Orai1表达特点及NFAT核聚集基础上，我们拟构建高静水压细胞模型模拟高血压状态，应用免疫共沉淀、激光共聚焦、膜片钳、Transwell等方法，研究STIM1/Orai1之间的作用对高血压状态下CASMCs钙内流及增殖、迁移的影响，应用信号通路抑制剂及基因沉默技术探讨calcineurin-NFAT信号对STMI 1/Orai 1的调控作用，以期揭示高压负荷诱导CASMCs功能变化的机制，为冠心病的防治提供理论依据。

背景：高血压是冠状动脉收缩功能障碍的主要危险因素之一，但其发病机制尚不清楚。.目的：研究高血压大鼠冠状动脉收缩功能变化规律，并探讨其相关的机制。.方法：采用多功能肌无力仪测量动脉环收缩。从SHR和wistar大鼠冠状动脉分离血管平滑肌细胞（VSMCs），用钙离子探针fluo-4/AM测定细胞内钙离子浓度。westernblotting检测信号通路相关蛋白。.结果：与wistar大鼠相比，5-HT和CaCl2诱导的SHR冠状动脉收缩作用明显减轻。5-HT2A受体阻滞剂沙格雷酯能完全消除5-HT引起的冠状动脉收缩，磷脂酶Cβ（PLCβ）抑制剂U73122也能显著抑制5-HT的反应。Rho相关蛋白激酶抑制剂Y27632、PKC抑制剂rottlerin、Cav1.2阻断剂硝苯地平和TRPC抑制剂SKF96365对SHR和wistar大鼠5-HT诱导的冠状动脉收缩有剂量依赖性抑制作用，但对SHR的抑制作用均减弱。此外，SOC在冠状动脉平滑肌细胞中引起明显的Ca2+内流，而在冠状动脉中SOC介导的收缩非常轻微。同时发现SHR冠状动脉血管平滑肌细胞5-HT2AR、IP3R、Cav1.2蛋白表达及PKC活性明显降低，stim1和Orai1明显升高。.结论：在5-HT诱导下5-HT2A受体及其下游信号通路介导的钙通道导致SHR冠状动脉收缩减少。