TMEM16A和ClC-3 Cl-通道参与脑血管重构的内在相互关联的分子机制及药物干预的研究

Under the NNSFC foundation support, we have found that Chloride channel is a new target for the blocking cerebrovascular remodeling. Although both of TMEM 16A Ca2+ activated and ClC-3 volume regulated Cl- channels evoked a decrease in the cytoplasma Cl- level ([Cl-]i), these channels mediated the cerebrovascular remodeling in reverse manner, suggesting that there is an interaction between TMEM 16A Ca2+ activated and ClC-3 volume regulated Cl- channels in the development of cerebrovascular remodeling. In this study, we would like to use spontaneously hypertensive rat two kidney two clip hypertensive rat, DoCa- salt hypertensive TMEM16A knockout and ClC-3 knockout mice for the investigating the interaction of two channels in the development of cerebrovascular remodeling, underling mechanisms and the kinas of sensitivity to the [Cl-]i change by ClC-3 channel, with patch clamp single channel recording, image and biomolecular techniques. Also we would like to determine the influence of statin-treatment. This study will provides a very important data to identify that chloride channel is a new target for the development the new drug for the treatment of cerebrovascular remodeling and stroke, and to further explain the molecular mechanism of chloride channels effect on cerebrovascular remodeling.

在前国家自然科学基金重点项目的资助下,已发现Cl-通道是一有效阻遏脑血管重构的新靶点。TMEM16A Ca2+激活与ClC-3容积调节Cl-通道均使[Cl-]i下降，但参与脑血管重构的方式刚好相反, 提示在高血压脑血管重构中，TMEM16A与ClC-3 Cl-通道相互间有内在关联。本项目拟在ClC-3敲除及TMEM16A敲除DOCA-盐高血压小鼠，SHRSP及双肾双夹易卒中高血压大鼠上，采用斑片钳离子通道记录, 荧光影象测定胞浆Cl- 及分子生物学等技术,进一步探讨TMEM16A与ClC-3 Cl-通道在脑血管重构中的内在关联机制；寻找ClC-3 Cl-通道降低[Cl-]i激活的Cl-敏感蛋白激酶，其信号通路，及与脑血管重构的关系;他汀类药物干预后的影响；为进一步揭示Cl-通道在脑血管重构作用的分子基础，为防治高血压脑血管重构及脑卒中等并发症的新药研发提供新靶点奠下基础。

探讨TMEM16A Ca2+激活Cl-通道与ClC-3容积调节性Cl-通道在高血压脑血管重构中的相关作用及内在的关联； 寻找ClC-3容积调节性Cl-通道相偶联的[Cl-]i降低所激活的Cl-敏感性蛋白激酶，其参与的信号通路，以及与脑血管重构的关系。. 研究显示，TMEM16A Cl-通道通过抑制外基质沉积，平滑肌细胞迁移, 超微结构变异以及改善血管内皮功能障碍, 改善脑血管重构。TMEM16A 与 ClC-3，在TMEM16A knock-in小鼠Ang II引起脑血管重构过程中的ClC-3表达没有明显改变， 它们之间也没有免疫共沉淀， 说明TMEM16A Ca2+激活与ClC-3容积调节性Cl-通道在高血压脑血管重构中没有直接的相互作用。 在脑血管重构发生过程中，TMEM16A和ClC-3 Cl-通道是受CaMKⅡ、endophilinⅡ及integrinβ3通过不同机理的调节，致使它们以相反的方式参与脑血管重构。. 在低渗刺激可促进WNK1磷酸化， 激活的WNK1使PI3K, AKT磷酸化，CyclinD1及CyclinE表达增加，p21，及p27表达减少，促进细胞周期从G1向S期转化，从而导致血管平滑肌细胞增殖。低渗通过激活WNK1抑制线粒体通路从而保护细胞凋亡。 . 研究工作为进一步揭示Cl-通道在脑血管重构的作用分子基础，并为防治高血压脑血管重构及脑卒中等并发症的新药研发提供新的靶点奠下了基础。