模拟失重大鼠脑动脉血管平滑肌细胞L-型Ca2+通道昼夜节律的改变及其作用机理

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. L-type voltage-dependent calcium (CaL) channels are highly expressed in vascular smooth muscle cells (VSMCs). It is believed that CaL channels play an essential role in the regulation of vascular function and structure. Our previous work has reported that simulated microgravity induced enhanced contraction and hypertrophic adaptation in rat cerebral arteries. Furthermore, we also demonstrated that simulated microgravity increased the function of CaL channel not only in daytime (12 h light) but also in nighttime (12 h dark) as compared with the control. These results suggested that the circadian regulation of CaL channels has changed in cerebral arterial remodeling of simulated microgravity rats. However, the precise mechanisms remain to be determined. In the present study, we want to investigate the role of circadian regulation of CaL channels in simulated microgravity rats with electrophysiology, molecular biology, culture cell, 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 the zeitgeber medicine for contermeasures to ensure the health and efficiency for 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脑血管失调的一个重要因素，其发生机理目前仍不甚清楚，也缺乏有效的对抗措施。L型电压依赖性钙通道(CaL）在血管平滑肌细胞（VSMCs）上广泛分布，对血管的功能与结构起着重要的调控作用。我们前期工作已证实模拟失重可引起大鼠脑动脉收缩功能增强、结构发生肥厚性重建，且脑动脉VSMCs膜上CaL通道功能在白天(12 h光照)与夜间（12 h黑暗）时均显著增强，提示CaL通道昼夜节律的改变可能参与模拟失重大鼠脑动脉功能与结构的重建过程，但其作用机理却不清楚。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CaL通道昼夜节律的改变机理及其在模拟失重大鼠脑动脉重建中的作用。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，并希望找到受时因子药物作为对抗措施，以保证航天员的健康与工作效率。

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脑血管失调的一个重要因素，其发生机理目前仍不甚清楚，也缺乏有效的对抗措施。L型电压依赖性钙通道(CaL）在血管平滑肌细胞（VSMCs）上广泛分布，对血管的功能与结构起着重要的调控作用。本研究证实，1）模拟失重可引起大鼠心脑血管功能发生似昼夜节律的紊乱，具体表现为：正常大鼠的心率、收缩压与舒张压均呈现出昼低夜高的振荡性改变，而模拟失重可引起大鼠日间与夜间心率显著增加，而收缩压与舒张压显著降低，且日间与夜间心血管功能的振荡幅度显著缩小。同时，模拟失重也可引起大鼠脑动脉血管紧张度似昼夜节律紊乱（即日间与夜间振荡幅度显著缩小）。2) 模拟失重大鼠脑血管功能似昼夜节律的紊乱涉及脑动脉L-Ca2+通道似昼夜节律的紊乱，且外周与中枢节律基因（per-2、bmal1与dbp）均发生mRNA与蛋白水平的改变。通过膜片钳电生理记录与蛋白印迹分析等技术证实：模拟失重可增强大鼠脑动脉Ca2+通道日间与夜间电流密度与蛋白表达，但昼夜振荡幅度显著减小；但通过qPCR分析发现模拟失重大鼠脑动脉Ca2+通道在转录水平的似昼夜节律变化不明显。通过蛋白印迹与qPCR分析，发现可模拟失重可改变大鼠脑动脉（外周时钟）与视交叉上核SCN（中枢时钟）节律基因（包括Per-2/Bmal1/dbp）的mRNA与蛋白表达的似昼夜节律特征。3）miRNA-103是介导模拟失重大鼠脑动脉L-Ca2+通道昼夜节律特征的关键信号：我们对所有已报道的以L-Ca2+通道为靶分子的miRNA进行筛选，在模拟失重大鼠脑动脉中发现miRNA-103的昼夜振荡变化最明显，继而将miRNA-103转染到培养的平滑肌细胞系中，发现激活miRNA-103（mimic）可显著抑制L-Ca2+通道的蛋白表达和昼夜节律特征；而抑制miRNA-103（inhibitor）可显著增强L-Ca2+通道的蛋白表达和昼夜节律特征。总之，本项目通过电生理膜片钳、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发现模拟失重大鼠脑动脉似昼夜节律的改变涉L-Ca2+通道昼夜节律的改变机理及其miRNA-103对L-Ca2+通道昼夜节律特征的调控作用。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，并希望找到受时因子药物作为对抗措施，以保证航天员的健康与工作效率。