血管平滑肌细胞LRRC8A Cl-通道的生物学特性与功能及与ClC-3 Cl-通道的关系和药物干预的研究

For a long time， volume regulated Cl- channel (VRCC) on the molecular identification is kept in debate. We have confirmed that ClC-3 is an effective component of VRCC in vascular smooth muscle cells, and participates in cerebral vascular smooth muscle cell proliferation, apoptosis and remodeling. Recent scholars have put forward LRRC8A is an effective component of VRCC. But not confirmed on different cells. LRRC8A features and functions are not known in vascular smooth muscle cells. To this end, we intend to work on the basis of the original, through LRRC8A knockout mice in vivo experiment, gene silencing and mutation in vitro, using patch clamp single ion channel recording, dynamic determination of the fluorescence images of Cl- transmembrane movement and molecular biology technology, to explore the biological characteristics of LRRC8A Cl- channel in vascular smooth muscle cells , the relationship between ClC-3 volume regulated and LRRC8A Cl- channels, and LRRC8A channel regulated signal pathway; to investigate whether the LRRC8A-Cl- channel involves into cell proliferation, apoptosis and cerebrovascular remodeling, as well as intervention of drugs. In order to reveal the biological characteristics and functions of LRRC8A in vascular smooth muscle cells, and provide new scientific basis and clues for LRRC8A as a new target for the prevention and treatment of hypertensive cerebral vascular remodeling and stroke.

容积调节Cl- 通道 (VRCC) 的分子基础一直存在着争论, ,我们证实ClC-3 是血管平滑肌细胞(VSMCs) VRCC的有效组成成份,参与脑血管平滑肌细胞增殖,凋亡及脑血管重构. 近有学者提出LRRC8A 是VRCC的有效组成成份。但是在不同的细胞上仍有争论。对血管平滑肌细胞上LRRC8A特性和功能一无所知。本项目拟在原有基础上，通过LRRC8A基因敲除鼠整体与基因沉默及突变等离体实验，采用斑片钳, 荧光影像测定单细胞Cl-跨膜运动及分子生物学等技术，探讨LRRC8A Cl-通道在血管平滑肌细胞上的生物学特性, 与ClC-3容积调节Cl-通道之间关系；LRRC8A-Cl-通道是否干预细胞增殖与凋亡参与脑血管重构，及药物对之干预作用。为揭示LRRC8A在血管平滑肌细胞的生物学特性及LRRC8A作为新靶点寻求防治高血压脑血管重构及脑卒中新药提供新的科学依据和线索。

本项目研究主要是解决脑血管平滑肌上LRRC8A的生物学特性以及对脑血管重构的影响。研究工作在离体细胞水平及整体动物（包括血管平滑肌特异性过表达LRRC8A动物模型）进行。研究发现LRRC8A是容积调节性Cl-通道的分子基础之一，它与另一个容积调节性Cl-通道的分子之一ClC-3存在交叉活性。.LRRC8A通过降低胞内Cl-水平激活Cl-敏感蛋白激酶WNK1，后者进一步激活PI3K-AKT通路，促进细胞周期因子cyclinE、D的表达，及细胞周期从G1向S期转变，导致细胞增殖。同时通过线粒体通路组织细胞凋亡。通过以上机制促进了高血压脑血管重构。.与此同时的研究，我们还发现细胞外低Cl-可激活ClC-5依赖的外向整流Cl-电流及降低胞内Cl-水平。虽然LRRC8A和ClC-5 Cl-通道是不同特性的Cl-通道，但它们均通过降低胞内Cl-水平激活WNK1从而促进脑血管重构。.这些发现可为防治脑血管疾病新药提供新线索和思路。