神经干细胞微囊泡抑制H2O2 诱导DRG 神经元氧化应激损伤机制研究

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 rootganglion, 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-tubulin Ⅲ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Annexin Ⅴ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400 μg/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cleavedcaspase3,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。

Research on Mechanisms of Neural Stem Cell Microvesicles Inhibit Oxidative Stress Injury in H2O2-Induced DRG Neurons

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 H2O2 group (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.