microRNA在人参皂甙Rd缺血性脑保护中的作用机制研究

Ischemic stroke is a high mortality disease caused by angiostenosis or vascular occlusion that reduce the focal blood perfusion and ultimately followed by cerebral impairment or infarct. The efficacy of Ginsenoside Rd (GSRd) in treating ischemic stroke has been evaluated in our pilot clinical trial. However, its underlying neuroprotective mechanisms remain largely unknown. We have found that the hippocampal levels of endogenous microRNA-21 and 34a increased significantly and such increase consistent with the infarct level of the brain tissue. Thus, we raised the hypothesis that miR-21 and 34a are involved in the ischemic brain impairments. However, the compositions of hippocampi are very much complicated and the miRs are coupled with many target genes, whether and how do these miRs involve in the neuroprotective effects of GSRd remain unknown. In the current study, the bioinformatics, neuropharmacological, and behavioral techniques will be used to investigate the dynamical changes of hippocampal miR-21 and 34a levels and the target cells and genes during the neuroprotective effect of GSRd. Then, the post-transcriptional regulation of target genes by miR within the hippocampi will be investigated with the gain and loss function study. The purpose of the current study is to offer promising new targets for ischemic stroke.

缺血性脑卒中是由于血管狭窄或闭塞阻断血流而引起局部脑组织损伤或坏死，具有高致残致死率。我们既往评估了人参皂甙Rd用于治疗缺血性脑卒中的疗效，但其具体作用机制不清。我们的预实验发现缺血海马组织内miRNA-21和miRNA-34a水平显著升高，并且与缺血范围和程度一致，提示它们可能参与缺血性脑损伤过程，但海马神经细胞构成复杂，且miRNA的靶基因众多，对其是否以及如何参与人参皂甙Rd的缺血性脑保护仍不清楚。本课题拟应用miR分子生物学、生物信息学、神经药理学和形态学方法观察海马miR-21和34a分子在人参皂甙参与的缺血性脑保护过程中的动态变化及参与此过程的细胞（神经元和胶质细胞）类型，确定其靶基因；进而研究抑制或增强海马神经细胞miR分子功能对缺血性脑损伤的转录后调控机制，预期将为开发新的治疗缺血性脑卒中的药物及选择有效的治疗策略提供新的干预靶点。

1. 建立了SD大鼠大脑中动脉结扎（MACO）的脑缺血模型.选取280-300g 成年雄性SD 大鼠进行实验，采用线栓法建立NACO模型，MACO造模2h，再灌注24 h后大鼠脑组织出现显著坏死。.2. microRNA芯片检测.提取MCAO后不同时间点（1h、24h和7d）对照和模型组动物损伤区、损伤周围区域脑组织，交公司进行microRNA芯片检测，利用miRBase预测miR的靶分子，利用MiRTarBase数据库和软件分析MCAO损伤相关miR的网络联系。根据芯片结果，选取miR34a和miR21作为目标分子进行后续研究。.3.MACO后不同时间点不同脑区内miR34a和miR21的表达变化. 利用实时定量RT-PCR，观察到MACO术后24小时海马、纹状体和丘脑的miR-21和34a较对照组明显增高，结果具有显著统计学差异，该变化在海马区域尤为显著。MACO模型术后24h海马组织 miR-34a分子和miR-21分子水平显著升高，并持续到术后7天。.4. 不同剂量人参皂甙（GSRd）对MACO的保护作用.MACO前给予不同剂量的GSRd，对MACO造成的缺血性脑损伤具有显著的保护作用。.目前已发表SCI论文一篇Wei D, Wan Q, Li L, Liu YH, Wang YG, Zhang GY. MicroRNAs as Potential Biomarkers for Diagnosing Cancers of Central Nervous System: a Meta-analysis. Mol Neurobiol. 2014 Aug 1. 撰写SCI论文一篇。