内皮损伤致血栓形成与KLF2分子调控相关性及益气活血方干预作用的研究

气虚血瘀证患者存在内皮细胞受损等改变，KLF2转录因子对血管内皮具有重要的保护作用，那么用于治疗气虚血瘀证的益气活血方是否通过KLF2调控途径保护血管内皮、抗血栓形成呢？本项目在阐明KLF2表达与体内外血栓形成密切相关的基础上，应用逆转录-多聚酶链合反应、基因获得以及基因敲除技术探讨KLF2表达水平对于内皮功能紊乱致血栓形成事件的价值；应用酶联免疫吸附法检测血栓形成时小鼠体内炎性因子的表达水平，探讨KLF2的调节作用；利用抗KLF2单抗或可溶性KLF2/IgG1封闭小鼠血栓形成模型体内的KLF2通路，或利用KLF2基因敲除小鼠，研究KLF2调控通路的阻断作用对于内皮功能失调及血栓形成的影响，探讨其作用机制，并观察益气活血方的干预作用。本项目的完成将阐明KLF2在内皮功能紊乱致血栓形成机制中的作用，并为临床应用益气活血方治疗血栓性疾病提供理论依。

Krüppel-like factor 2 has an important role in the protection of the biological function of the vascular endothelium. On the other hand, the patients with syndrome of blood stasis due to qi deficiency tend to suffer from the damage of endothelial cells, and so on. We hypothesize that Yiqihuoxue Decoction may be used for the treatment of endothelial dysfunction and thrombus formation via KLF2 regulatory pathway. First, we focus on the close relationship between KLF2 expression levels and thrombus formation in vitro and in vivo. Then, using reverse transcription polymerase chain reaction and gain- and loss-of-function approaches, we identify the value of KLF2 on the thrombosis events related to the endothelial dysfunction. For the next step, enzyme linked immunosorbent assay is applied for the observation of the expression of cytokine accompanied with the formation of thrombus in mice, and the regulatory role of KLF2 is elucidated. Finally, the anti-KLF2 mAb or soluble KLF2/IgG1 is used respectively to block in vivo the KLF2 pathways in mice, or KLF2 knock-out mice are used, to explore the effects of KLF2 pathway on endothelial dysfunction and thrombus formation, as well as to assess its mechanism underlying the antithrombotic role. On the basis of above, Yiqihuoxue Decoction is added to observe its intervention on the thrombosis because of endothelial dysfunction. In a word, the accomplishment of this grant will be to clarify the mechanism of KLF2 in thrombus formation caused by endothelial dysfunction and to provide a theoretical basis for the clinical application of Yiqihuoxue Decoction in the treatment of thrombotic diseases.