T型Ca2+通道在模拟失重大鼠脑动脉血管平滑肌细胞“表型转化”中的作用及间断性人工重力对其干预研究

Cerebrovascular remodeling has been regarded as one of important factors which may induce postspaceflight cardiovascular dysfunction. However, the mechanism remains unknown and there are still no effective countermeasures. Our previous work has demonstrated that simulated microgravity induced the hypertrophic remodeling in rat cerebral arteries with the phenotypic switching of cerebral VSMCs. Recently, we found that T-type Ca2+ channels participated in the phenotypic switching of cerebral VSMCs during simulated microgravity, the signal pathway included the transcription factor (Myocadin and SRF), Ca2+/calmodulin-dependent protein kinase II (CaMKII), and microRNA-103. However, the intermittent artificial gravity could prevent these changes, but the underling mechanisms remain to be determined. In the present study, we want to investigate the role of T-type Ca2+ channel in the phenotypic switching of cerebral VSMCs in simulated microgravity rats and the prevention of intermittent artificial gravity with electrophysiology, molecular biology, culture cells, and cell transformation techniques in vivo and in vitro. Our study may provide a novel mechanism for cerebrovascular remodeling during real/simulated microgravity. We also want to find a new drug target for countermeasure to ensure the health and efficiency of astronauts.

航天失重环境所引起的脑动脉功能与结构重建是航天员“心血管功能失调”的重要因素，其发生机理目前仍不甚清楚，也缺乏有效的对抗措施。我们前期工作已证实模拟失重可引起大鼠脑动脉血管平滑肌细胞（VSMCs）由“收缩表型”向“合成表型”转化，且脑动脉发生肥厚性重建。我们最新工作表明T型Ca2+通道可参与调控模拟大鼠脑动脉VSMCs表型转化过程，其机制可能涉及转录因子（Myocadin与SRF）、钙调蛋白激酶（CaMKII）与microRNA-103的变化，而间断性人工重力可有效防止脑动脉VSMCs表型转化。本项目拟采用电生理膜片钳、分子生物学方法、血管体外培养与细胞转染等技术从动物整体、器官、细胞与基因等多个水平探讨T型Ca2+通道在模拟失重大鼠脑动脉VSMCs表型转化中的作用以及间断性人工重力对其产生何种干预。本项目可对航天失重环境下脑动脉的重建机制提出新的思路，并希望找到新的药物靶点作为对抗措施。

航天失重环境所引起的脑动脉功能与结构重建是航天员“心血管功能失调”的重要因，其发生机理目前仍不甚清楚，也缺乏有效的对抗措施。血管平滑肌细胞（vascular smooth muscle cells, VSMCs）由“收缩表型”向“合成表型”的转换是血管重建的关键性起始步骤，但模拟失重大鼠脑动脉VSMCs是否发生表型转换，其分子机制是什么，国内目前外还没有报道。本研究采用电生理膜片钳、分子生物学、细胞转染、双荧光报告酶基因等技术方法证实以下四个发现。1）模拟失重可引起大鼠脑动脉VSMCs由“收缩表型”向“合成表型”转换：电镜超微结构显示尾部悬吊大鼠脑动脉VSMCs的肌纤维含量减少、线粒体、内质网等细胞器含量增多；尾部悬吊大鼠脑动脉SM-MHC、SM--actin、SM22等“收缩表型”分子标志物的表达显著降低，而OPN与PCNA等 “合成表型”分子标志物的表达却显著增高。2）T型CaV3.1通道的激活是调控模拟失重大鼠脑动脉VSMCs表型转换的关键步骤：尾部悬吊大鼠脑动脉T型CaV3.1的表达与功能均显著增加；以小干扰RNA（SiCaV3.1）或特异性阻断剂Mibefradil阻断T型CaV3.1通道可明显抑制A7r5平滑肌细胞的表型转换。3）miR-137可作为上游信号直接靶向作用于T型CaV3.1通道，而活化后的T型CaV3.1通道可分别激活下游的calcineurin/NFATc3转录因子核转位信号与线粒体融合/分裂信号，进而诱导模拟失重大鼠脑动脉VSMCs的表型转换；4）间断性人工重力间断性人工重力（每日23 h尾部悬吊+1 h恢复正常体位）能够通过下调T型CaV3.1及其下游信号通路，从而抑制模拟失重所诱导的大鼠脑动脉VSMCs表型转换。本研究阐明了T型CaV3.1通道能是失重/模拟失重调控脑动脉血管重建的关键信号因子，而且间断性人工重力也可通过这一分子靶点发挥脑血管的保护干预作用。本研究对揭示血管的生理与病理性重建提出了新的见解，也对航天员的医学保障具有重要的参考价值。