ClC-3氯通道调控血栓形成及其机制研究

Thrombosis is the most common pathological change in blood and circulation system , ion channels play a key role in the regulation of thrombus formation. Our previous experiments confirmed that ClC-3 chloride channel mediated TNF-α-induced endothelial cell chloride movement, regulation of the inflammatory response of endothelial cells involved in the development of cardiovascular disease. Preliminary experiments showed that platelets had ClC-3 protein , platelet agonist induced expression of platelets ClC-3 protein upregulation.This indicated ClC-3 chloride channel may be involved in thrombus formation. After siRNA transfection inhibited the expression of ClC-3, platelet agonists such as thrombin induced GPIIb and PECAM-1 expression was significantly decreased. On this basis, this study uses ClC-3 knockout mice to observe the role of ClC-3 on ferric chloride-induced carotid artery thrombosis model. And we further observed the effect of ClC-3 on platelet activation and aggregation in the process of thrombosis in vivo. The main object was to research the role of ClC-3 in the thrombosis process and the possible mechanism, to provide the assessment of ClC-3 chloride channel a new laboratory evidence for prevention and treatment of cardiovascular and cerebrovascular diseases.

血栓形成是血液循环系统最常见的病理变化，离子通道在调控血栓形成过程中发挥关键的作用。我们前期实验证实ClC-3氯通道是容积调节性氯通道的分子基础，该通道介导TNF-α诱导的血管内皮细胞氯运动，调控内皮细胞炎症反应，参与心脑血管疾病发生发展。预实验结果显示，血小板中有ClC-3蛋白表达，血小板激动剂诱导血小板中ClC-3蛋白的表达上调。提示ClC-3氯通道可能参与血栓形成过程。siRNA转染抑制ClC-3表达后，thrombin等激动剂诱导的血小板GPIIb/IIIa和PECAM-1表达显著下降。在此基础上，本研究拟采用ClC-3基因敲除小鼠，观察ClC-3氯通道敲除对三氯化铁诱导颈动脉栓塞模型中血栓形成的影响。并进一步采用ClC-3基因敲除小鼠观察在体情况下ClC-3氯通道对血栓形成过程中血小板活化和聚集的影响。本项目旨在为评估ClC-3在防治心脑血管疾病中的作用提供新的实验室依据。

血小板活化及其参与的血栓形成是严重的病理状态之一，已有研究表明钙钠等阳离子调控血栓形成。然而ClC-3氯通道及其介导的氯离子运动是否调控血小板活化及血栓形成目前并不明确。本研究从ADP激活的血小板中ClC-3的表达及胞内氯离子浓度变化出发，观察抑制氯通道与低氯对ADP诱导的血小板活化的影响，并进一步探讨低氯对ADP促进血小板活化的机制。本研究首先确认ClC-3氯通道在血小板中的表达以及血小板激动剂ADP、Thrombin和Collagen对血小板ClC-3表达的影响。其次，发现阻断氯通道或激活氯通道分别抑制或促进ADP诱导的血小板活化标志物PAC-1及p-selectin的表达、血小板粘附及聚集。最后，阐明低氯促进ADP诱导的血小板活化不是通过Lyn/PI3K/Akt活化途径，而是通过Gai/cAMP/PKA抑制途径实现的。此外，我们还在脑梗病人中验证血小板高表达ClC-3，而血清氯离子水平降低，表明监测血小板ClC-3表达及血清氯离子浓度在急性脑梗塞的发病及预后具有重要意义。