TLR4/miR-199a-3p轴调控缺血性脑卒中后血管新生的机制研究

Ischemic stroke is an important cause of death and disability in human, and angiogenesis after stroke is beneficial to the prognosis of patients. Based on our previous studies, we proved that in TLR4 knockout mice, angiogenesis in the ischemic brain hemisphere was reduced; in addition, based on our previous microRNA microarray and in situ hybridization, we found that miR-199a-3p significantly increased in the ischemic hemisphere of TLR4 knockout mice; and the number of vessels and the levels of angiogenesis-related proteins in miR-199a-3p knockout mice were higher than those in wild type mice after middle cerebral artery occlusion. Therefore, we hypothesize that the TLR4/miR-199a-3p axis plays an important role in the regulation of angiogenesis after stroke. Furthermore, bioinformatics analysis, primary cultured endothelial cells in vitro, and animal model in vivo will be used to clarify the mechanism of how TLR4 regulates miR-199a-3p and how miR-199a-3p inhibits angiogenesis. The aim of the current study is to improve stroke prognosis and to promote neurological recovery via regulation of angiogenesis.

缺血性脑卒中是人类致死致残的重要原因，而卒中后的血管新生有利于患者预后。基于前期研究，我们发现TLR4敲除小鼠脑梗死半球血管新生数量少于野生型小鼠；进一步结合前期microRNA芯片与原位杂交结果，我们发现TLR4敲除小鼠脑梗死半球出现miR-199a-3p显著上升；利用miR-199a-3p敲除小鼠进行大脑中动脉闭塞模型造模，发现敲除鼠新生血管数量与血管新生相关蛋白的表达均高于野生型。由此提出假说：TLR4/miR-199a-3p轴在卒中后血管新生中起重要作用。同时为了进一步明确其机制，本研究拟通过生物信息学分析、原代培养内皮细胞体外实验、动物模型体内实验求证：TLR4如何调控miR-199a-3p，以及miR-199a-3p抑制血管新生的机制。旨在为临床上改善卒中患者预后与促进神经功能恢复提供新的思路与理论依据。

项目组前期研究发现抑制Toll样受体4在脑梗死早期能够通过减轻炎症反应，起到神经保护作用，但对脑梗死后期的影响尚未明确。基于文献调研，我们发现长链非编码RNA SNHG1在脑梗死后上调，通过生物信息学分析和文献调研，我们进一步发现SNHG1启动子区存在TLR4下游转录因子NF-κB结合位点，同时SNHG1上存在miR-199a-3p结合位点，而miR-199a-3p敲除可显著增加梗死周边带血管数量。基于上述认识，我们开展本项研究，结果发现，脑缺血后在神经元中存在TLR4的异常激活，且与miR-199存在交互作用。本研究进一步探索了TLR4引起脑缺血后期血管新生的具体通路及调控机制，为脑缺血慢性期的治疗探索了潜在靶点，具有一定的临床转化意义。