miR-126-5p通过DLK1/Akt/eNOS通路促进血管新生治疗慢性缺血性脑血管病的作用与机制

Promoting the angiogenesis in chronic ischemic brain tissue can improve cerebral blood perfusion and reduce the risk of stroke, but the mechanism for regulating cerebral angiogenesis is not clear. Our previous study found that miR-126-5p can promote the proliferation and tube formation of human brain microvascular endothelial cells under normoxia in vitro. At the same time, PI3K/Akt/eNOS was involved in the formation of neonatal cerebral blood vessels as a downstream pathway. Combining with that miR-126-5p may participate the molecular regulation of angiogenesis directly through inhibiting DLK1. We hypothesize that miR-126-5p can targetedly inhibitie DLK1 expression, and then reduce the inhibition of PI3K/Akt pathway from DLK1 , and then up-regulate the expression of eNOS in endothelial cells, thereby promote angiogenesis. This project is a continuation of the previous research, and it is intended to use dual luciferase reporter to determine the target mRNA of miR-126-5p, and to use mRNA interference as an intervention method. Through cell biology experiments in vitro , combined with regional angiogenesis experiments on chronic cerebral ischemia rats models in vivo, we are planning to determine the effect and molecular mechanism of miR-126-5p in promoting cerebral angiogenesis, to explore the downstream regulatory pathway of DLK1, and to find new therapeutic targets for the improvement of clinical efficacy of chronic ischemic cerebrovascular diseases.

促进慢性缺血脑组织内新生血管的形成能够改善脑血流灌注并降低脑卒中风险，但调控脑血管新生的机制尚不明确。我们前期研究发现：miR-126-5p可在体外促进常氧状态下人脑微血管内皮细胞的增殖成管，以及PI3K/Akt/eNOS作为下游通路参与调控大鼠脑内血管新生。结合DLK1可能作为miR-126-5p的靶基因参与血管新生的调控，我们提出假设：miR-126-5p可通过靶向抑制DLK1的表达，降低后者对PI3K/Akt通路的抑制，进而上调内皮细胞eNOS的表达，最终促进血管新生。本项目是前期研究的延续，拟采用双荧光素酶报告明确miR-126-5p的下游靶基因，并以mRNA干扰为干预手段，通过体外细胞学实验以及慢性脑缺血大鼠局部脑血管新生的体内研究，来明确并验证miR-126-5p促进脑血管新生的作用和分子机制，同时探索DLK1的下游调控通路，为慢性缺血性脑血管病临床疗效的提高寻找新的治疗靶点。

促进慢性缺血脑组织内新生血管的形成能够改善脑血流灌注并降低脑卒中风险，但调控脑血管新生的机制尚不明确。我们课题组在前期研究发现miR-126-5p可在体外促进常氧状态下人脑微血管内皮细胞的增殖成管，以及PI3K/Akt/eNOS作为下游通路参与调控大鼠脑内血管新生，结合DLK1可能作为miR-126-5p的靶基因参与血管新生的调控，对miR-126-5p促进脑血管新生的机制进行进一步研究。通过对双侧颈动脉结扎慢性脑缺血大鼠模型（2VO）实施颞肌贴敷手术（EMS），并将miR-126-5p agomir/antagomir转染贴敷在脑表面的颞肌组织，并通过siRNA、慢病毒载体等方式调控下游基因的表达，明确了miR-126-5p促进缺血脑组织内皮细胞增殖的下游靶基因为DLK1，并提示了miR-126-5p可能是通过靶向抑制DLK1基因，进一步通过PI3K/Akt通路，发挥促进血管再生的作用，即分子通路miR-126-5p→DLK1→PI3K/Akt→eNOS。有助于我们课题组下一步对包含过表达miR-126-5p的BMSCs的水凝胶脑贴片的研发，有希望在未来10年实现临床转化，为慢性缺血性脑血管病的间接搭桥治疗提供更好的疗效。