节律基因Clock对小鼠凝血纤溶作用的调控机制研究

The development of Chronobiology gives us a new way of thinking on the regulation mechanism of thrombosis, which promotes the study of the regulation factors in coagulation and fibrinolysis system. Domestic and foreign studies proved that some important factors in thrombosis have an inextricably link to circadian rhythm and circadian genes. Our pre-experiment also found that the downregulation of Clock gene could cause a reduction of thrombus in male mice, and screened the potential target proteins which are involved in the regulation of Clock to coagulation-fibrinolysis system. This study is to investigate how the main circadian gene Clock function with the coagulation-fibrinolysis system in mice, to search for its signal pathways in the regulation of thrombosis. It could study in front the regulatory pathway of circadian Clock Gene in coagulation-fibrinolysis system, provide a new perspective to the regulation mechanism of thrombosis, and also provide a experimental basis for the prevention and treatment of this pathological processes.

时间生物学的发展为血栓形成的调控机制研究打开了新的思路，推动了对凝血和纤溶系统调节因素的研究。国内外研究均证明在血栓形成中具有重要作用的凝血相关因子与近日节律和节律基因有着密不可分的联系。本实验室也在前期实验发现节律基因Clock的表达下调能够降低小鼠血栓的形成，并找到了Clock基因调控凝血纤溶系统的潜在靶蛋白。本项目就是研究节律基因Clock对小鼠凝血纤溶系统的作用方式，并且寻找其调控血栓形成发展的信号通路，既能率先研究节律钟基因Clock在凝血纤溶系统中的调控通路，同时又从新的角度和途径对调控血栓形成的机制作出新的研究和诠释，也为该病理过程的防治提供实验依据。

本项目在对候选蛋白进行研究后，最终只锁定了一个候选蛋白Alpha-1-antitrypsin。通过研究我们发现Clock基因对Alpha-1-antitrypsin的调控是通过非常规NF-KB信号通路完成的，Alpha-1-antitrypsin会随着Clock的敲降而升高，其下游因子NE（中性粒细胞弹性蛋白酶）的表达则会降低，但仍具有节律性；而Alpha-1-antitrypsin的上调还可以通过抑制Active Protein C来抑制PAI-1的表达；之后的血浆生物标记物筛选及细胞水平验证实验还发现，Clock基因的下调还可以引起Fibronectin（Fn1）表达的升高，这可以导致对血小板聚集抑制的增加。这提示Clock敲降引发的纤溶增强，可能是通过对血小板聚集的影响、对非常规NF-KB信号通路调节对炎症反应的影响、以及Alpha-1-antitrypsin对Active Protein C的抑制来抑制PAI-1的表达等方面来实现的。在对血浆生物标记物筛选结果进行功能聚类分析后发现，除了凝血、纤溶、补体等相关的生物进程和信号通路的富集外，还存在明显的抗感染免疫、和毒素运输的富集；而对小鼠全血的血培养也证实，Clock基因敲降小鼠的全血比对照组小鼠的全血会更早的长出更多的菌落；提示Clock基因敲降后引起的小鼠纤溶增强可能与细菌感染增加有关。但这部分猜想还有待实验的进行进一步验证。