星形胶质细胞外泌体miRNA-485调控脑缺血-再灌注损伤的作用及机制研究

The role and effect of astroglia in cerebral ischemia-reperfusion injury (I/R injury) has not been clarified. In the preliminary study, we found that the expression of miRNA-485 in astroglia exosome was significantly increased during hypoxia injury, which could inhibit p53 pathway and apoptosis in primary neuron, and alleviated neuronal damage in vitro. Therefore, we hypothesis that astroglia plays an important role in cerebral I/R injury: the up-regulation of miR-485 in the astroglia derived exosome could generate the protective effects against the cerebral I/R injury via inhibiting the p53 pathway in vivo. To demonstrate the hypothesis, this project intends to confirm the role of astroglia derived exosome miR-485 in distal MCA occlusion (dMCAO) model in rats firstly; then to reveal the mechanism on the protective effects of astroglia derived exosome miR-485 against cerebral I/R injury injury by inhibiting the p53 in Oxygen and Glucose Deprivation (OGD) model in vitro and cerebral ischemia model in vivo; finally to explore the cerebral I/R injury may activate HIF-1α pathway in astroglia and induce the miR-485 expression in the astroglia derived exosome. This research is expected to find the new mechanism of astroglia exosome miR-485 on the regulating the cerebral I/R injury by inhibiting the p53 pathway, and to provide new insights and targets for the treatment of cerebral I/R injury.

脑缺血-再灌注损伤时星形胶质细胞所起的作用尚未明确。我们前期体外实验发现在缺氧损伤时，星形胶质细胞外泌体miRNA-485表达显著增高，可抑制神经元p53通路并减轻神经元损伤程度。因此我们推测脑缺血-再灌注损伤时星形胶质细胞存在重要作用：其分泌的外泌体内miR-485表达增高，通过抑制神经元p53通路调控脑缺血-再灌注损伤。为明确这一假说，本项目拟首先在大鼠脑缺血模型中确认星形胶质细胞外泌体miR-485的调控作用；然后在体内外脑缺血模型中揭示星形胶质细胞外泌体miR-485通过抑制p53通路调控脑缺血-再灌注损伤并产生保护作用及具体机制；最后探索脑缺血时星形胶质细胞可通过激活HIF-1α而上调其外泌体内miR-485的表达水平。本课题有望明确星形胶质细胞外泌体miR-485通过抑制p53通路调控脑缺血-再灌注损伤的全新机制，为脑缺血-再灌注损伤的治疗提供新的思路及目标靶点。

本研究在前期研究基础上，首先确认了星形胶质细胞外泌体miR-485对脑缺血-再灌注损伤（脑I/R损伤）的调控作用：本课题第一部分及第二部分中通过体外原代培养小鼠原代星形胶质细胞，并建立体外脑I/R损伤模型；超速离心法分离纯化星形胶质细胞外泌体，通过第二代测序技术对星形胶质细胞来源的外泌体进行miRNA转录组测序，发现星形胶质细胞外泌体miR-485参与了脑缺血再灌注损伤的过程，并可靶向作用于神经元；同时应用miR-485 mimics可减轻体内外脑I/R损伤，而miR-485 inhibitor可加重体内外脑I/R损伤，证实miR-485对脑I/R损伤的调控作用。..其次，探索脑I/R损伤时星形胶质细胞外泌体miR-485表达水平增高、通过抑制p53通路产生保护作用的机制：本课题第三部分中采用双荧光素酶报告基因分析miR-485的具体作用靶点，表明p53为其下游靶点；进一步将星形胶质细胞外泌体与体外培养的小鼠原代神经元共孵育，表明外泌体可以靶向神经元发挥作用；在此基础上，构建神经元特异性p53过表达转基因小鼠，原代培养神经元，miR-485 mimic介导神经元p53转录及表达下调，逆转p53过表达引起的神经元损伤及神经元凋亡。..第三，明确HIF-1α通路的激活介导了脑I/R损伤时星形胶质细胞外泌体miR-485表达增高的机制：本课题第四部分中在体内脑缺血-再灌注损伤模型中发现梗死区域脑组织Hif-1α表达上调；应用病毒载体侧脑室注射，小鼠脑组织Hif-1α表达水平进一步升高，可使得miR-485表达升高，p53转录及表达下降，降低脑梗死面积及脑水肿，抑制神经元凋亡，减轻脑I/R损伤。..因此我们得出结论，脑缺血-再灌注损伤时星形胶质细胞存在重要的作用：星形胶质细胞内HIF-1α表达增高，从而上调其分泌的外泌体中miR-485的表达水平，作用于靶细胞（神经元），通过抑制p53通路而对缺血损伤产生保护作用。本课题阐明脑缺血损伤时星形胶质细胞外泌体的保护作用及机制，可在一定阶段向临床进行转化，开发相关的药物及作用靶点，具有较好的应用前景。