2-ME调控HIF-1α信号通路抑制硬脑膜动静脉瘘血管生成的作用机制研究

硬脑膜动静脉瘘(DAVF)的病理基础是硬脑膜动静脉之间形成新生血管，导致动静脉短路。我们曾发现低氧诱导因子（HIF-1α）介导的血管生成对DAVF发病起关键作用，是极具前景的治疗靶标。2-甲氧雌二醇（2-ME）能通过抑制HIF-1α逆转大鼠DAVF形成，但分子机制尚不明确。本项目前期已构建人脐静脉内皮细胞（HUVEC）低氧模型，并已利用RNA芯片筛选2-ME抑制HIF-1α的信号通路，拟采用基因敲减（shRNA）或过表达的方法对其中的关键调控因子加以验证，以免疫共沉淀、双分子互补实验（BiFC）和构建突变体验证上游调控因子与HIF-1α的相互作用。再采用经我们创新改良的DAVF大鼠模型，颅内注射搭载关键调控因子RNA/shRNA的慢病毒，在体内验证2-ME抑制DAVF中HIF-1α激活的信号通路，据此构建2-ME抑制DAVF中HIF-1α的调控网络，为应用2-ME治疗DAVF提供理论基础。

Angiogenesis is the pathological basis of dural arteriovenous fistula (DAVF), leading to arteriovenous shunt in dura. Our previous studies revealed that hypoxia-inducible factor (HIF) -1α mediated angiogenesis caused by hypoxia plays a key role in DAVF formation, and thus it is a potential therapeutic target in DAVF. Meanwhile, our studies also show that 2-methoxyestradiol (2-ME) can remarkably suppress dural angiogenesis mediated by HIF-1α. However, detailed upstream signal pathway and regulatory network are still unclear. In this project, we have firstly used human umbilical vein endothelial cell (HUVEC) for screening candidate upstream factors regulated by 2-ME through RNA microarray. Then we will check each pathway by inhibiting or overexpressing candidate regulatory factors in vitro. Furthermore, we will examine the interaction of those molecules by co-immunoprecipitation, bimolecular fluorescence complementation (BiFC) and mutant tecniques. In the last stage, we will verify each signal pathway which can suppress HIF-1α regulated by 2-ME in vivo, through a modified DAVF rat model and local lentivirus intervention. Finally we will try to build regulatory network of 2-ME among HIF-1α and upstream factors, which can provide theoretical basis for further clinical trials.