外泌体在M1型小胶质细胞诱发神经元损伤中的作用

Role of exosomes in neuronal injury induced by M1 microglia

目的评价外泌体在M1型小胶质细胞诱发神经元损伤中的作用。方法将生长良好的BV2小胶质细胞加入脂多糖100 ng/ml和干扰素-γ20 ng/ml诱导小胶质细胞极化为M1表型。收集M1型小胶质细胞上清液,提取外泌体。将生长良好的N2a细胞采用随机数字表法分为4组(n=24):对照组(C组)、M1型小胶质细胞组(M组)、外泌体组(E组)和外泌体抑制剂+M1型小胶质细胞组(G+M组)。C组常规培养24 h;M组加入M1型小胶质细胞上清液培养24 h;E组加入M1型小胶质细胞源性的外泌体培养24 h;G+M组于M1型小胶质细胞加入外泌体抑制剂GW4869培养24 h后,取上清液,加入到N2a细胞中培养24 h。采用CCK-8法检测N2a细胞活力,流式细胞术检测细胞凋亡率,qRT-PCR法检测Bcl-2和Bax的mRNA表达,Western blot法检测凋亡相关基因Bcl-2和Bax的表达。结果与C组比较,其余3组细胞活力降低,细胞凋亡率升高,Bcl-2及其mRNA表达下调,Bax及其mRNA表达上调(P<0.05);与M组比较,G+M组细胞活力升高,细胞凋亡率下降,Bcl-2及其mRNA表达上调,Bax及其mRNA表达下调(P<0.05),E组和M组上述指标差异无统计学意义(P>0.05)。结论M1型小胶质细胞可通过外泌体介导神经元损伤。

Objective To evaluate the role of exosomes in neuronal injury induced by M1 microglia.Methods Liposolysaccharide 100 ng/ml and interferon-γ(IFN-γ)20 ng/ml were added to well-growing BV2 microglia to induce the polarization of microglia into M1 phenotype.Cell supernatant of M1 microglia was collected and M1 microglia exosomes(M1-exo)were extracted with exosome kit.The well-growing N2a cells were divided into 4 groups(n=24 each)using a random number table method:control group(group C),M1 microglia group(group M),exosome group(group E),and exosome inhibitor+M1 microglia group(group G+M).The cells in group C were conventionally cultured,the cells in group M were cultured with the supernatant of M1 microglia for 24 h,and the cells in group E were cultured with M1 microglia-derived exosomes for 24 h.In G+M group,exosome inhibitor GW4869 was added,M1 microglia were incubated for 24 h,then the supernatant was collected and added to N2a cells,and the cells were incubated for 24 h.Cell viability of N2a cells was measured by the cell counting kit 8 assay,cell apoptosis rate was determined by flow cytometry.The expression of apoptosis-related genes Bcl-2 and Bax mRNA was detected by quantitative real-time-polymerase chain reaction,and the expression of apoptosis-related genes Bcl-2 and Bax protein was detected by Western blot.Results Compared with group C,the cell viability was significantly decreased,the apoptosis rate was increased,the expression of Bcl-2 protein and mRNA was down-regulated,and the expression of Bax protein and mRNA was up-regulated in the other three groups(P<0.05).Compared with group M,the cell viability was significantly increased,the apoptosis rate was decreased,the expression of Bcl-2 protein and mRNA was up-regulated,and the expression of Bax protein and mRNA was down-regulated in group G+M(P<0.05).There was no significant difference in the above indexes between group E and group M(P>0.05).Conclusions M1 microglia can mediate neuronal injury via exosomes.