TRPP2-STIM1相互作用：脑缺血再灌注损伤新机制研究

Stroke is a serious harm to the safety of life and refractory disease. Cerebral vasospasm induced by Ca2+ overload in cerebral vascular smooth muscle cell is one of the important reasons for ischemic stroke and reperfusion injury. However, Ca2+ channel antagonist does not have good outcome on ischemic stroke and reperfusion injury in cilinical treatment. Recent studies found that TRP channels are widely expressed in vascular smooth muscle cells and mediate intracellular Ca2+ overload and spasms. However, Ca2+ channel antagonist has no effect on TRP channels. We firstly found that TRPP2 and STIM1 form a signal complex in Ca2+ store membrane of vascular smooth muscle cell. Ca2+ store depletion caused by the opening of TRPP2 quickly activates STIM1. Then STIM1 mediates Ca2+ influx by store-operated Ca2+ entry. Thus we predict that brain tissue releases blood vessel contract factors to induce intracellular Ca2+ overload, spasms and blood supply decrease via activating TRPP2-STIM1 complex during cerebral ischemia reperfusion. Therefore, we plan to use TRPP2-STIM1 as a target, TRPP2 conditional knockout mouse and cerebral ischemia reperfusion animal modal to study: (1)the function of TRPP2-STIM1 in cerebral ischemia reperfusion injury; (2)the change of TRPP2-STIM1 complex in hypertension, the effect on hypertensive cerebral ischemia reperfusion injury and the value of the complex in clinical teatment. The study will provide a novel pathological mechanism of cerebral ischemia reperfusion injury and a novel target for clinical treatment.

脑卒中是严重危害生命安全的难治性疾病，脑血管平滑肌钙超载引起痉挛缺血是脑缺血再灌注损伤的重要原因之一。但临床钙拮抗剂治疗缺血再灌注损伤效果不佳，近年研究发现，血管平滑肌也广泛表达钙通透的TRP通道，可引起钙超载和血管痉挛，而钙拮抗剂对TRP通道无效。我们首次发现血管平滑肌钙库膜上TRPP2通道与STIM1结合成复合物，TRPP2开放可迅速激活STIM1,介导钙库操纵钙内流、血管痉挛。所以预测脑缺血再灌注时分泌的血管收缩因子通过激活TRPP2-STIM1复合物引起平滑肌钙超载、脑动脉痉挛、血供降低。所以拟以TRPP2-STIM1为靶点，通过血管平滑肌TRPP2条件性敲除小鼠和脑缺血再灌注模型研究TRPP2-STIM1：(1)在脑缺血再灌注损伤中的作用；(2)在高血压时的改变和高血压脑缺血再灌注损伤中的作用及临床治疗中的价值。该研究将为脑缺血再灌注损伤提供新的病理机制，为临床治疗提供新的靶点。

项目背景：脑卒中是严重危害人类健康和生命安全的常见的难治性疾病，根据各种急性脑血管病发生的病因、病理及不同表现，脑卒中包括出血性和缺血性两类，缺血引起的脑损伤不仅发生在组织灌注不足时，更重要的发生在灌注恢复期，这种现象称为脑缺血再灌注（ cerebral ischemia reperfusion, CIR）损伤。研究表明，CIR 时，缺血缺氧等因素可导致脑组织分泌大量的内源性血管收缩因子。近年很多资料表明，脑 CIR 时分泌的内源性血管收缩因子可通过信号转导激活瞬时受体电位（transient receptor potential, TRP）通道，研究发现TRPP家族TRPP2可与钙库操纵钙内流（Store-operated Ca2+ entry, SOCE）中的重要分子STIM1（stromal interaction molecule 1, STIM1）相 互作用形成钙信号复合物，介导SOCE。.研究内容：TRPP2-STIM1相互作用在高血压动物脑动脉平滑肌中的表达水平以及其对SOCE的影响，同时检测TRPP2-STIM1相互作用在高血压动物大脑 CIR 损伤中的作用。.主要结果：通过研究，我们发现TRPP2 离子通道与激动剂引起的血管收缩有密切关系， 而且 TRPP2 通道蛋白在高盐诱导的高血压大鼠大血管和阻力血管平滑肌上表达显著增多，可能是高盐诱导高血压大鼠血管张力增加和发病的一个重要因素；采用自发性高血压大鼠（SHR）和SD对照大鼠，检测ET-1引起的脑基底动脉钙库操纵钙内流导致的血管收缩。结果显示：高血压大鼠的ET-1引起的脑基底动脉钙库操纵的钙内流导致的血管收缩显著增强；另外通过荧光共振能量转移技术鉴定出TRPP2和STIM1是通过TRPP2的N端和STIM1的C端相互作用形成复合物。采用TRPP2平滑肌条件性敲除小鼠，通过血管张力实验发现激动剂引起的TRPP2血管平滑肌收缩功能显著减弱；同理，敲低STIM表达可以显著抑制ATP引起的SOCE。另外，我们通过测序结果发现高盐能引起血管平滑肌细胞中与增殖密切关联的FYN基因的mRNA显著改变，进一步Western实验也证实了该发现。.研究意义：该研究为临床治疗CIR损伤提供理论基础，为药物筛选提供潜在治疗靶点。