内皮祖细胞源性外泌体对氧糖剥夺-复氧复糖神经元损伤的影响

Effects of endothelial progenitor cell-derived exosomes on neuronal injury induced by oxygen-glucose deprivation and restoration

目的评价内皮祖细胞源性外泌体对氧糖剥夺-复氧复糖神经元损伤的影响。方法培养HT22小鼠海马神经元,采用随机数字表法分为3组(n=30):对照组(C组)、氧糖剥夺-复氧复糖组(OGD/R组)和氧糖剥夺-复氧复糖+内皮祖细胞源性外泌体组(OGD/R+EXO组)。C组细胞正常培养,OGD/R组采用无糖无血清DMEM培养基在94%N_(2)-1%O_(2)-5%CO_(2)混合气体培养6 h进行氧糖剥夺,随后更换正常培养基进行复氧复糖24 h。OGD/R+EXO组于氧糖剥夺-复氧复糖造模前24 h在培养基中加入20μg/ml内皮祖细胞源性外泌体。采用免疫荧光染色法鉴定内皮祖细胞,Western blot法、透射电镜、纳米颗粒追踪分析法鉴定外泌体。采用CCK-8法检测神经元活力,ELISA法测定MDA含量和SOD活性,TUNEL染色法检测神经元凋亡率,Western blot法检测凋亡相关蛋白的表达。结果培养细胞为内皮祖细胞,成功提取内皮祖细胞源性外泌体。与C组相比,OGD/R组和OGD/R+EXO组神经元活力降低,MDA含量升高,SOD活性降低,神经元凋亡率升高,Bax表达上调,Bcl-2表达下调,cleaved-caspase-3/caspase-3比值升高(P<0.05);与OGD/R组相比,OGD/R+EXO组神经元活力升高,MDA含量降低,SOD活性升高,神经元凋亡率降低,Bax表达下调,Bcl-2表达上调,cleaved-caspase-3/caspase-3比值降低(P<0.05)。结论内皮祖细胞源性外泌体可减轻氧糖剥夺-复氧复糖神经元损伤,与抑制氧化应激和神经元凋亡有关。

Objective To evaluate the effects of endothelial progenitor cell(EPC)-derived exosomes on neuronal injury induced by oxygen-glucose deprivation and restoration(OGD/R).Methods HT22 neurons of mice were cultured and divided into 3 groups(n=30 each)using a random number table method:control group(C group),OGD/R group and OGD/R plus EPC-derived exosome group(OGD/R+EXO group).Cells in group C were cultured in normal atmosphere.In group OGD/R,the cells were exposed to 94%N_(2)-1%O_(2)-5%CO_(2) for 6 h in glucose-and serum-free DMEM medium,followed by 24 h restoration of O_(2) and glucose in the normal medium.In group OGD/R+EXO,20μg/ml EPC-derived exosomes were added to the culture medium at 24 h before developing the model.EPCs were identified by immunofluorescence staining.Exosomes were identified by Western blot,transmission electron microscopy(TEM)and nanoparticle tracking analysis(NTA).Cell viability was measured by CCK-8 assay,the levels of malondialdehyde(MDA)and superoxide dismutase(SOD)were determined by enzyme-linked immunosorbent assay.The neuronal apoptosis was detected by TUNEL staining,and the apoptosis rate was calculated.The expression of Bax,Bcl-2,caspase-3,and cleaved caspase-3 was determined by Western blot,and cleaved caspase-3/caspase-3 ratio was calculated.Results The cultured cells were EPCs,and EPC-derived exosomes were successfully extracted.Compared with group C,the cell viability was significantly decreased,the content of MDA was increased,the activity of SOD was decreased,the apoptosis rate was increased,the expression of Bax was up-regulated,the expression of Bcl-2 was down-regulated,and the ratio of cleaved-caspase-3/caspase-3 was increased in group OGD/R and group OGD/R+EXO(P<0.05).Compared with group OGD/R,the cell viability was significantly increased,the content of MDA was decreased,the activity of SOD was increased,the apoptosis rate was decreased,the expression of Bax was down-regulated,the expression of Bcl-2 was up-regulated,and the ratio of cleaved-caspase-3/caspase-3 was decreased in group OGD/R+EXO(P<0.05).Conclusions EPC-derived exosomes can reduce OGD/R-induced neuronal injury,which is related to inhibition of oxidative stress and neuronal apoptosis.