背景与目的： 缺血性卒中具有发病率高、死亡率高和致残率高的特点，其治疗一直是神经病学的重点和难点。缺血预处理，或缺血预适应(ischemicpreconditioning，IPC)，是缺血性脑损伤的一种重要治疗手段。但是，缺血预处理诱导神经保护作用的本质及调控机制尚未阐明，值得深究。MicroRNA(miRNA)是一类内源性单链非编码RNA分子，其长约18-25个核苷酸。miRNA通过与相应的靶mRNA结合对其进行降解或抑制其翻译，参与多种生物学过程。近年来研究提示miRNA介导的调节参与神经元的发育、塑性以及分化，在缺血性脑卒中的发生发展过程中发挥着重要的作用。外泌体(exosome，Exo)是机体内细胞分泌的微小囊泡，其直径在30-100 nm之间。外泌体被视为一种载体，可运输miRNA到相应的靶细胞，影响受体细胞的功能。越来越多的证据证明，外泌体在细胞间信息传导方面起到了非常重要的作用。基于这些理论，我们尝试探究缺血预处理的神经保护作用是否是通过外泌体所介导的细胞间信息传导所引起。为此，我们设计了本课题，这一课题旨在为缺血预处理机制研究提供新的思路，也为缺血性卒中的治疗提供新的干预靶点。 方法： 1.体外分别培养原代神经元细胞、原代星形胶质细胞、小胶质细胞系(BV2)。建立3种细胞的缺血预处理模型，分别收集3种细胞缺血预处理48 h后的条件培养基，将获得的条件培养基分别加入原代培养的神经元中，孵育24 h后对神经元进行2.5 h糖氧剥夺(oxygen-glucose deprivation，OGD)损伤，使用细胞计数试剂盒、细胞活性染色试剂盒和TUNEL染色评估不同细胞的条件培养基对神经元的影响。 2.通过超高速离心和外泌体提取试剂盒的方法提取条件培养基中的外泌体，利用透射电子显微镜、Western Blot对提取的外泌体进行验证。利用免疫荧光的技术检测星形胶质细胞分泌的外泌体是否可以被神经元摄取。将提取的外泌体加入到神经元中孵育24h后对细胞进行2.5h的糖氧剥夺损伤，通过细胞计数试剂盒、细胞活性染色和TUNEL染色评估星形胶质细胞缺血预处理后分泌的外泌体对神经元的影响。 3.通过高通量测序检测缺血预处理前后星形胶质细胞分泌的外泌体中miRNA的差异表达，差异较大的miRNA进行实时荧光定量PCR鉴定。 4.使用miR-92b-3p抑制剂和miR-92b-3模拟物下调或上调神经元中miR-92b-3p的表达后，对细胞进行2.5 h的糖氧剥夺损伤。通过细胞计数试剂盒、细胞活性染色和TUNEL染色评估miR-92b-3p是否是缺血预处理神经保护的关键作用因子。 5.使用双荧光素酶报告基因和Western Blot的技术验证miR-92b-3p的靶基因。建立小鼠脑缺血预处理模型和大脑中动脉阻塞(middle cerebral artery occlusion， MCAO)模型，在给予小鼠MCAO的同时将miR-92b-3p拮抗剂或miR-92b-3p激动剂注射到小鼠脑室内下调或上调小鼠脑内miR-92b-3p的表达。使用神经功能缺损评分(modified Neurological Severity Scores，mNSS)和贴纸去除实验评估不同处理组小鼠神经功能情况，使用TTC染色评估小鼠大脑梗死体积，使用TUNEL染色评估小鼠脑内细胞凋亡情况，使用Western Blot评估miR-92b-3p对PTEN／Akt／mTOR信号通路的调节。 结果： 1.星形胶质细胞缺血预处理后的条件培养基可以减轻神经元糖氧剥夺后的损伤，而神经元和小胶质细胞条件培养基对神经元不存在保护作用。 2.星形胶质细胞条件培养基内富含外泌体，并且这些外泌体可以被神经元所摄取。神经元摄取外泌体后可以减轻由糖氧剥夺引起的细胞损伤。 3.与对照组相比，缺血预处理后星形胶质细胞分泌的外泌体中miR-92b-3p的表达明显升高。 4.下调神经元中miR-92b-3p的表达会拮抗由星形胶质细胞条件培养基构成的神经保护作用，上调神经元中miR-92b-3p的表达会产生与星形胶质细胞条件培养基相比拟的神经保护作用。 5.PTEN为miR-92b-3p的靶基因。与单纯MCAO组相比给予小鼠MCAO损伤之前先给予小鼠脑缺血预处理模型会减轻小鼠MCAO术后神经功能的损害，降低脑梗死体积，减轻细胞凋亡，下调脑组织中PTEN蛋白的表达，增加p-Akt和m-TOR的表达。使用miR-92b-3p拮抗剂下调小鼠脑内miR-92b-3p的表达会增加脑内PTEN蛋白的表达，降低p-Akt、p-mTOR蛋白的表达，同时会加重小鼠MCAO后的神经功能损害，增加脑梗死体积，增加脑内细胞的凋亡，拮抗由缺血预处理引起的神经保护作用。相反，使用miR-92b-3p激动剂上调小鼠脑内miR-92b-3p的表达可以产生与缺血预处理相比拟的神经保护作用。 结论： 星形胶质细胞缺血预处理后分泌的外泌体可以减轻脑缺血后的损伤，其作用机制可能与外泌体介导的miR-92b-3p旁分泌参与调节PTEN／Akt／mTOR信号通路有关。 关键词：缺血预处理；外泌体；miRNA；PTEN／Akt／mTOR信号通路

 Background and Objective: Ischemic stroke is a central nervous system disease with high incidence, high mortality and high morbidity. The treatment of ischemic stroke has always been the emphasis and difficulty to medical researchers. Ischemic preconditioning (IPC) is a viable strategy to treat ischemic cerebral injury. However, the mechanisms underlying the IPC-induced neuroprotection remain unclear. MicroRNA (miRNA) is a class of endogenous singlestranded non-coding RNA (containing about 18-25 nucleotides). MiRNAs participate in a variety of biological processes by binding to the 3'UTR of their target mRNA to interfere with their translation. In recent years, studies have suggested that miRNA is involved in the development, plasticity and differentiation of neurons and plays an important role in the development and progression of ischemic stroke. Exosomes are membrane vesicles with a diameter of 30-100 nm secreted by many cell types. Exosomes can transport and deliver various miRNA to recipient cells and can modify cell function. Numerous studies have been shown that exosomes played an important role in intercellular communication. Accordingly, we try to explore whether the neuroprotective effect of IPC is mediated by exosomes. This study will help to underpin the theory of ischemic preconditioning and may provide novel potential approach for the management of ischemic stroke. Methods: 1.Primary cultured astrocytes, neurons and BV2 cell line. Cells were subjected to oxygen-glucose deprivation (OGD, astrocytes for 3 h, neurons for 30 min and BV2 for 2 h) to build the cell ischemic preconditioning model. Neurons were cultured with or without the conditioned medium for 24 h and subjected to 2.5 h of OGD. Cell counting kits, LIVE/DEAD BacLight Viability kits and TUNEL staining to assess the effect of conditioned medium on neurons. 2.Extraction of exosomes in conditioned medium by ultracentrifuge and exosome isolation kits. Transmission electron microscope and Western Blot were used to confirm the presence of exosomes in the conditioned medium. Immunofluorescence staining was used for detecting whether exosomes from astrocytes were taken up by neurons. Neurons were cultured with or without exosome for 24 h and subjected to 2.5 h of OGD. Cell counting kits, LIVE/DEAD BacLight Viability kits and TUNEL staining to assess the effect of exosomes from astrocyte conditioned medium on neurons. 3.MiRNA sequence was carried out in exosomes before and after astrocytes ischemic preconditioning. Significantly differentially expressed miRNAs were identified by real time PCR. 4.Neurons were subjected to 2.5 h OGD after down-regulated or up-regulated the expression of miR-92b-3p by miR-92b-3p inhibitor or miR-92b-3p mimic. Cell counting kits, LIVE/DEAD BacLight Viability kits and TUNEL staining to assess whether exosome was required and sufficient to confer neuroprotection in cultured neurons. 5.Dual luciferase reporter assays and Western Blot were used to verify the target mRNA of miR-92b-3p. We used 15 min middle cerebral artery occlusion (MCAO) surgery to induce IPC model in mice. Inhibited or overexpressed the expression of miR-92b-3p by intraventricular injection of miR-92b-3p inhibitor or miR-92b-3p mimic. Modified Neurological Severity Scores (mNSS) and adhesive removal test were used to evaluate the neurological function of mice after MCAO surgery. TTC staining and TUNEL staining were used to assess the infraction volume and apoptosis after MCAO surgery. Western Blot was used to detect the effect of miR-92b-3p on PTEN/Akt/mTOR signaling pathway. Results: 1.Culture medium from ischemic preconditioned astrocytes but not neurons and BV2 ameliorated OGD-induced neuron injury. 2.Conditioned medium from astrocytes were enriched with exosomes. Exosomes from astroglial conditioned medium were taken up by neurons and protected neurons from OGD-induced injury. 3.The expression of miR-92b-3p in exosomes released by ischemic preconditioned astrocytes was significantly increased. 4.Downregulated the expression of miR-92b-3p in neurons resisted the neuroprotective effect of astrocyte conditioned medium. However, overexpression of miR-92b-3p with miR-92b-3p mimic exerted similar protection effects on the cell survival compared with astrocyte conditioned medium. 5.PTEN was a target mRNA of miR-92b-3p. Deletion of miR-92b-3p aggravated the neurological impairment and increasd the infraction volume and TUNEL-positive cells after MCAO surgery. Overexpression of miR-92b-3p exert similar protection effects on the mice. The neuroprotection of ischemic preconditioning was regulated by miR-92b-3p/PTEN/Akt/mTOR pathway. Conclusion: Exosomes released by ischemic preconditioning astrocytes exert neuroprotection in vitro and in vivo. The mechanism of the neuroprotection of exosome maybe associated with miR-92b-3p mediated PTEN/Akt/mTOR pathway. Key words: ischemic preconditioning; exosome; miRNA; PTEN/Akt/mTOR pathway