神经干细胞微囊泡抑制H_(2)O_(2)诱导DRG神经元氧化应激损伤机制研究  

Research on Mechanisms of Neural Stem Cell Microvesicles Inhibit Oxidative Stress Injury in H_(2)O_(2)-Induced DRG Neurons

作　　者：唐彬 万长春[1] 宗寿洋 胡嘉波[2] TANG Bin;WAN Chang-chun;ZONG Shou-yang;HU Jia-bo(Jinhu County People’s Hospital,Jiangsu Jinhu 211600,China;School of Medicine,Jiangsu University,Jiangsu Zhenjiang 212013,China)

机构地区：[1]金湖县人民医院,江苏金湖211600 [2]江苏大学医学院,江苏镇江212013

出　　处：《现代检验医学杂志》2022年第4期159-164,共6页Journal of Modern Laboratory Medicine

摘　　要：目的探究神经干细胞微囊泡(neural stem cell microvesicles,NSC-MVs)对H_(2)O_(2)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H_(2)O_(2)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,H_(2)O_(2)组神经元活力明显抑制。当H_(2)O_(2)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,H_(2)O_(2)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H_(2)O_(2)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H_(2)O_(2)诱导DRG神经元氧化应激损伤,发挥神经保护作用。Objective To investigate the effect and mechanism of neural stem cell microvesicles(NSC-MVs)on oxidative stress injury in H_(2)O_(2)-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 ofβ-tubulinⅢ.The oxidative stress injury model was established in DRG neurons induced with H_(2)O_(2) and the concentration was determined.After the pretreatment with NSC-MVs,the cell viability of neurons was detected by MTT,AnnexinⅤand 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 H_(2)O_(2) group compared with control group.When H_(2)O_(2) concentration was 25,50,100 and 200μmol/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 400μg/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 H_(2)O_(2) group compared with the control group(P<0.05),neuronal apoptosis in NSC-MVs pretreatment group was obviously inhibited compared with the H_(2)O_(2) group(P<0.05).Western blotting results showed that compared with the H_(2)O_(2) 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 H_(2)O_(2)-induced DRG neuron oxidative stress damage and play a neuroprotective role.

关 键 词：神经干细胞 微囊泡 背根神经节 氧化应激 凋亡 

分 类 号：R446.8[医药卫生—诊断学]