过表达miR-124-1基因的骨髓间充质干细胞外泌体调控小胶质细胞M2型极化对脑卒中的影响

Effect of exosomes derived from mesenchymal stem cells with miR-124-1 overexpression on rat model of stroke by regulating M2 polarization of microglia

目的探讨骨髓间充质干细胞(bone marrow derived mesenchymal stem cells,BMMSCs)外泌体(exosomes,Exo)过表达miR-124-1基因(Exo/124-1)调控小胶质细胞(microglia,MG)的M2型极化对脑卒中的影响。方法分离培养大鼠BMMSCs,收集其Exo(BMMSCs-Exo),采用流式细胞术、Western blot与透射电子显微镜(transmission electron microscope,TEM)分别对其进行检测鉴定。30只大鼠简单随机分为假手术组(Sham组)、模型组(MCAO/R组)、Exo移植组(Exo组)、空病毒(Empty lentivirus,Elv)转染Exo移植组(Exo-Elv组)及Exo/124-1移植组(Exo/124-1组),每组6只。Sham组仅行假手术,其余各组均复制大脑中动脉栓塞/再灌注(middle cerebral artery occlusion and reperfusion,MCAO/R)模型。模型复制1 d与14 d后,各移植组动物于右侧脑室植入相应移植物,Sham组、模型组注入相同剂量生理盐水作对照。术后2 h及1、3、7、14、21、28 d,分别对各组动物行改良神经系统严重程度评分(modified neurological severity scores,mNSS)。三苯基四氮唑(triphenyl tetrazolium chloride,TTC)染色检测其梗死体积。在基因与蛋白水平分别检测各组28 d脑组织MG的M1型分子[肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)]、M2型分子(CD206)的表达情况。结果成功获取并鉴定了BMMSCs及其Exo。Exo/124-1显著表达miR-124-1。所有动物(假手术组除外)术后出现神经功能缺损。术后7~28 d,Exo/124-1组的mNSS明显低于MCAO/R组(P<0.05)与Exo组、Exo-Elv组(P<0.01);术后28 d,Exo/124-1组的脑梗死体积、TNF-α的表达明显小于MCAO/R组(P<0.01)与Exo组、Exo-Elv组(P<0.01),CD206的表达显著高于MCAO/R组(P<0.01)与Exo组、Exo-Elv组(P<0.01)。结论BMMSCs-Exo携带miR-124-1基因可能调控MG的M2型极化,抑制M1型介导的炎症反应,促进脑卒中大鼠神经功能的恢复。

Objective To investigate the effect of exosomes(Exo)derived from bone marrow mesenchymal stem cells(BMMSCs)overexpressing miR-124-1 by regulating M2 polarization of microglia(MG)on stroke.Methods Rat BMMSCs were isolated and cultured.Then BMMSCs-Exo were extracted,and flow cytometry,Western blotting and transmission electron microscopy(TEM)were used to identify the BMMSCs and BMMSCs-Exo,respectively.Thirty rats were randomized into sham operation group(sham group),model group(middle cerebral artery occlusion and reperfusion,MCAO/R),Exo transplantation group(Exo group),transplantation group of Exo transfected with empty lentivirus(Exo-Elv group)and transplantation group of Exo transfected with124-1 lentivirus(Exo/124-1 group),with 6 rats in each group.In 1 and 14 d after modeling,the corresponding agents were transplanted into the right lateral ventricle of rats in each transplantation group,while the rats of Sham group and MCAO/R group were given the injection of the same dose of normal saline.At 2 h and 1,3,7,14,21 and 28 d after modeling,modified neurological severity score(mNSS)was employed to evaluate neurologic deficit,and triphenyl tetrazolium chloride(TTC)staining was performed to measure cerebral infarct volume.The expression of TNF-α(M1 molecule)and CD206(M2 molecule)in the MG in the brain tissue at 28 d at both mRNA and protein levels were detected.Results BMMSCs and BMMSCs-Exo were successfully obtained and identified.MiR-124-1 was significantly expressed in Exo/124-1 rats.All rats(except those from the sham operation group)had neurological deficit after modeling.From 7 d to 28 d after modeling,the mNSS was significantly lower in the Exo/124-1 group than the MCAO/R group(P<0.05)and the Exo group and the Exo-Elv group(P<0.01).At 28 d after modeling,the infarct volume was notably smaller and the TNF-αexpression level was significantly lower in the Exo/124-1 group than the MCAO/R group(P<0.01)and the Exo group and Exo-Elv group(P<0.01),and the CD206 level was remarkably higher than the MCAO/R group(P<0.01)and the Exo group and Exo-Elv group(P<0.01).Conclusion BMMSCs-Exo carrying miR-124-1 may regulate the polarization of MG to M2 type,inhibit M1 type-mediated inflammatory response,and thus promote the recovery of neurological function in stroke rats.