短暂性脑缺血发作大鼠血清外泌体源性miRNA诊断标志物的筛选及功能验证

It is difficult to differentiate TIA(transient ischemic attack) from ischemic stroke within the time window of thrombolysis. Although TIA may be diagnosed within a restricted time window utilizing the latest imaging technologies, complexity and high cost of technologies limit their widespread use.A great number of serum markers are applied to the diagnosis and prognosis of many diseases for their characteristic of non-invasive,quick and economic,while diagnosic markers of exosome-derived miRNA for TIA have not been described. For this purpose, we intend to establish focal brain ischemia which is produced by the occlusion of the middle cerebral artery (MCAO) at different time points(5min,10min,2h) in rats. Usin deep sequencing and chip technology , we examine the global expression profiling of miRNAs. The miRNAs showed a consistent trend for change in the cerebrospinal fluid（CSF）and serum will be validated by qPCR technology and bioinformatics analysis to identify the potential serum exosome-derived miRNA biomarkers. This study will be capable to determine diagnosis for TIA quickly and efficiently, reducing the cost and promoting thrombolysis in acute ischemic stroke.

短暂性脑缺血发作（TIA）与缺血性脑卒中在溶栓时间窗内难以鉴别。不断发展的影像诊断技术，虽能在有限的治疗时间窗内证实TIA的诊断，但因其技术复杂和费用高昂而使得这些技术难以普及。血液标志物检测具有非创伤性、快速及成本低廉等优点而备受关注，并已有大量血液标志物被研究应用于疾病的诊断或预后判断等，然而针对TIA诊断的血液外泌体源性miRNA标志物迄今未见报道。为此，本项目拟建立不同时间点（5min,10min,2h）大脑中动脉闭塞的大鼠模型，应用深度测序及芯片技术对血清和脑脊液中miRNA的表达差异进行分析，筛选血清及脑脊液中表达趋势一致并与早期缺血损伤相关的关键miRNA，然后分离及鉴定出血清关键外泌体源性miRNA，通过qPCR技术和生物信息学分析对其进行表达及功能验证。本项目的实施将有助于快速有效地早期诊断TIA，提高溶栓治疗的开展率，降低溶栓治疗风险，具有重要的科学意义和应用价值。

目前尚未发现短暂性脑缺血发作（TIA）的血液诊断标志物。本项目通过建立不同时间点（5min,10min,2h）大脑中动脉闭塞的大鼠模型，应用深度测序及芯片技术对血浆中外泌体源性miRNA的表达差异进行分析，使用PCR技术及生物信息学分析筛选出在血浆与脑脊液中表达一致的外泌体源性miRNA，其中在缺血5分钟组中，血浆外泌体源性rno-miR-300-3p表达量的ROC曲线下面积AUC=0.970，在缺血10分钟组中，血浆外泌体源性rno-miR-122-5p表达量的ROC曲线下面积AUC=0.960，提示血浆外泌体源性rno-miR-300-3p和rno-miR-122-5p对大鼠TIA的诊断具有很高的潜在价值。未来需要在TIA患者中进一步验证。若验证成功，则这些血浆标志物有可能应用于TIA的临床诊断，帮助提高溶栓的治疗效率。