缺氧诱导的库操纵钙通道分子表达及相互作用——肺血管壁重塑的分子机制研究

Store-operated Ca2+ entry (SOCE) plays a very important role in the occurrence and development of pulmonary heart disease inudced by pulmonary artery hypertension. At present, two progresses have been suggested in regulation of SOCE that Orai1 contributes to Ca2+ release-activated Ca2+ (CRAC) channels while TRPC1 is the candidate of SOCs, and stromal interaction molecule 1(STIM1) is a Ca2+ sensor in the sarcoplasmic/endoplasmic reticulum. However, the exact composition of SOCs is not yet established. Here we will set up hypoxic pulmonary hypertension rat model and several kinds of pulmonary artery smooth muslce cell strains containing different SOC component by silencing selectively the expression of TRPC1, Orai1 and STIM1, and detect their expressions and interactions induced by hypoxia and analyze their effects on proliferation in rat pulmonary artery smooth muslce cells. Expected results will elucidate the molecular mechanism of native SOCs in pulmonary artery smooth muscle cells, which will not only advance understanding of regulation mechanism of SOCE but also offer the basis for clinical treatment of pulmonary artery hypertension induced by hypoxia.

库操纵的钙通道（SOCs）介导的钙内流（SOCE）在肺动脉高压引起的肺源性心脏病发生与发展过程中发挥重要作用。目前SOCE的调控机制取得了两大进展：其一，已确认Orai1为钙释放激活的钙通道组成单位，TRPC1为SOCs候选蛋白；其二，已明确基质相互作用因子（STIM1）为肌质网钙感受器。但SOCs确切的分子机制一直未能阐明。本项目拟通过缺氧性肺动脉高压动物模型与基因沉默获得含不同SOCs分子的肺动脉平滑肌细胞株（细胞模型）两种途径，从不同层面分析缺氧诱导的TRPC1，Orai1及STIM1的天然表达及相互作用对大鼠肺动脉平滑肌细胞增殖的影响。预计本项目的研究将揭示肺动脉平滑肌天然SOCs的分子机制。该机制的阐明不仅有助于加深对SOCE调控机制的理解，而且为缺氧性肺动脉高压的临床治疗提供理论依据。

库操纵的钙通道（SOCs）介导的钙内流（SOCE）与活性氧（ROS）在缺氧诱导的肺动脉高压发生与发展过程中均发挥重要作用。但SOCs确切的分子机制及其与ROS之间的关系一直未能阐明。本项目通过基因沉默获得含不同SOCs分子的肺动脉平滑肌细胞株，分析TRPC1，Orai1及STIM1的天然表达与相互作用对大鼠肺动脉平滑肌细胞SOCE的影响及其在缺氧诱导的SOCE变化中的作用，并进一步地探讨了缺氧诱导的ROS与SOCE之间的关系。结果显示，单独沉默TRPC1，Orai1或STIM1的表达均显著削弱SOCE，免疫共沉淀实验表明这三种蛋白质存在相互作用；缺氧可诱导TRPC1，Orai1及STIM1的表达，且显著抬高肺动脉平滑肌细胞SOCE；缺氧不仅增加肺动脉平滑肌细胞内ROS水平，也增加过氧化氢水平；用过氧化氢酶而非超氧阴离子清道夫超氧化物歧化酶显著降低SOCs通道分子的表达，并几乎完全清除缺氧诱导的SOCE；外源性过氧化氢可模拟缺氧诱导的肺动脉平滑肌细胞中SOC通道分子的相互作用变化。这些研究发现表明TRPC1，Orai1及STIM1是引起肺动脉平滑肌细胞SOCE的必需成分，缺氧诱导的ROS通过其转化产物过氧化氢增强的TRPC1，Orai1及STIM1的表达及其相互作用，继而抬高库操纵的钙内流。