【摘要】 目的 探究神经干细胞微囊泡（neural stem cell microvesicles, NSC-MVs）对H₂O₂诱导背根神经节（dorsal root ganglion, DRG）神经元氧化应激损伤的作用及机制。方法 超速离心提取NSC-MVs，并进行电镜和纳米颗粒示踪分析。原代培养大鼠DRG神经元，β-tubulinⅢ荧光染色。建立H₂O₂诱导DRG神经元氧化应激损伤模型，确定作用浓度。经NSC-MVs预处理，MTT（四唑盐）检测神经元活力，流式细胞术检测AnnexinⅤ和PI，蛋白质印迹检测凋亡相关蛋白cleaved caspase 3,cleaved caspase 9,Bax和Bcl-2的表达。结果 NSC-MVs在透射电镜下呈圆盘状，包膜完整，纳米颗粒示踪显示其粒径为50～450 nm。MTT结果显示，与对照组相比，H₂O₂组神经元活力明显抑制。当H₂O₂浓度为25,50,100和200μmol/L时具有显著性差异，细胞活力分别为84.4%,73.7%,69.8%和49.5%（F=127.7,P <0.01）。经100,200和400μg/ml的NSC-MVs预处理DRG神经元，细胞活力得到明显提升，分别为51.4%,67.4%和73.5%（F=49.47,P=0.023）。流式细胞术检测结果显示，与对照组相比，H₂O₂组神经元凋亡率显著上升（P <0.05）,NSCMVs预处理组细胞凋亡率明显下降（P <0.05）。蛋白质印迹结果显示，与H₂O₂组相比，NSC-MVs显著抑制cleaved caspase3,cleaved caspase 9和Bax蛋白表达（均P <0.05），上调Bcl-2蛋白表达（P <0.05）。结论 NSC-MVs能够抑制H₂O₂诱导DRG神经元氧化应激损伤，发挥神经保护作用。更多还原

【Abstract】 Objective To investigate the effect and mechanism of neural stem cell microvesicles（NSC-MVs） on oxidative stress injury in H₂O₂-induced DRG neuron. Methods NSC-MVs were extracted by ultracentrifugation and analyzed by electron microscopy and nanoparticle tracer. Rat dorsal root ganglion（DRG） neurons were cultured and identified by fluorescence staining of β-tubulin Ⅲ. The oxidative stress injury model was established in DRG neurons induced with H₂O₂ and the concentration was determined. After the pretreatment with NSC-MVs, the cell viability of neurons was detected by MTT, Annexin Ⅴ and PI in DRG neurons were detected by flow cytometry, the apoptosis-related protein expression of cleaved caspase 3, cleaved caspase 9, Bax and Bcl-2 were detected by western blotting. Results The NSC-MVs were disc-shaped with intact envelope under transmission electron microscope, the particle size was 50-450 nm by nanoparticle tracer analysis. MTT assay showed that DRG neuron viability was significantly inhibited in H₂O₂ group compared with control group. When H₂O₂ concentration was 25, 50,100 and 200 μmol/L, the cell viability was 84.4 %, 73.7 %, 69.8 % and 49.5% respectively（F = 127.7, P < 0.01）. The viability of DRG neurons pretreated with 100, 200 or 400 μg/ml NSC-MVs was significantly increased to 51.4 %, 67.4 % or 73.5 %（F=49.47,P =0.023）. Flow cytometry results showed that apoptotic rate increased remarkably in H₂O₂ group compared with the control group（P < 0.05）, neuronal apoptosis in NSC-MVs pretreatment group was obviously inhibited compared with the H₂O₂group（P < 0.05）. Western blotting results showed that compared with the H₂O₂ group, expressions of cleaved caspase 3, cleaved caspase 9 and Bax proteins were down-regulated（all P < 0.05）, while Bcl-2 was up-regulated in NSC-MVs-treated group（P< 0.05）. Conclusion NSC-MVs can inhibit H₂O₂-induced DRG neuron oxidative stress damage and play a neuroprotective role.更多还原