缺血预处理脑血管内皮细胞分泌的外泌体对氧葡萄糖剥夺神经元的保护作用

Exosomes derived from cerebral vascular endothelial cells after ischemic preconditioning protect neurons against oxygen-glucose deprivation-induced injury

目的:探讨缺血预处理(ischemic preconditioning, IPC)脑血管内皮细胞bEnd.3分泌的外泌体(exosome, Exo)对氧葡萄糖剥夺(oxygen and glucose deprivation, OGD)神经元的影响。方法:将bEnd.3暴露于OGD 3 h模拟体内IPC,复氧48 h后提取条件培养液中的外泌体(IPC Exo),采用蛋白质印迹分析和透射电镜方法进行鉴定Exo。将IPC Exo与原代培养的小鼠大脑皮质神经元共同孵育24 h,共聚焦显微镜观察Exo能否被原代培养的小鼠大脑皮质神经元摄取。将原代培养的小鼠大脑皮质神经元分为对照组、OGD组、OGD+IPC Exo(5μg/ml、10 μg/ml、20 μg/ml)组以及Sham OGD组(常氧条件培养的bEnd.3分泌的Exo进行处理),采用CCK-8和细胞存活/死亡检测试剂盒检测细胞活力。结果:透射电镜观察显示,bEnd.3培养液提取物呈现Exo典型形态,即直径30-100 nm的双凹圆盘状囊泡。蛋白质印迹分析显示,bEnd.3培养液提取物高度表达Exo标志物Alix和Tsg101。共聚焦显微镜观察显示,Exo可被原代培养的小鼠大脑皮质神经元摄取,摄取的Exo广泛分布于胞质和突触。与OGD组比较,加入10 μg/ml和20 μg/ml IPC Exo可显著提高神经元活力( P<0.05),而加入Sham Exo则无神经保护作用。 结论:IPC脑血管内皮细胞释放的Exo对OGD神经元具有保护作用。

Objective To investigate the effect of exosomes(Exo)secreted by brain vascular endothelial cell bEnd.3 after ischemic preconditioning(IPC)on neurons suffering from oxygen and glucose deprivation(OGD).Methods bEnd.3 was exposed to OGD for 3 h to simulate IPC in vivo.After 48 h of reoxygenation,the Exo(IPC Exo)in the conditioned medium were extracted and identified by Western blot and transmission electron microscopy.IPC Exo were incubated with primary cultured mouse cortical neurons for 24 h.Confocal microscope was used to observe whether Exo could be uptaked by primary cultured mouse cerebral cortical neurons.The primary cultured cortical neurons were divided into control group,OGD group,OGD+IPC Exo(5μg/ml,10μg/ml,20μg/ml)groups and sham OGD group(treated with Exo secreted by bEnd.3 cultured under normoxia conditions).The cell viability was detected by CCK-8 and cell survival/death detection kit.Results Transmission electron microscopy showed that the extract of bend.3 culture medium showed typical morphology of Exo,i.e.,a double concave disc-shaped vesicle with a diameter of 30-100 nm.Western blot analysis showed that the extract of bEnd.3 medium highly expressed Exo markers Alix and Tsg101.Confocal microscopy showed that Exo could be uptaked by primary cultured mouse cortical neurons,and the uptake of Exo was widely distributed in the cytoplasm and synapses.Compared with the OGD group,the addition of 10 and 20μg/ml IPC Exo could significantly increased the neuronal viability(P<0.05),while the addition of sham Exo had no neuroprotective effect.Conclusion Exo released by cerebral vascular endothelial cells after IPC have protective effect on neurons suffering from OGD.