骨髓间充质干细胞外泌体介导miR-124-1对小胶质细胞M2型极化调控的影响

Effect of miR-124-1 mediated by exosomes of bone marrow-derived mesenchymal stem cells on the regulation of transformation of M2 microglia

目的·探讨过表达miR-124-1基因的骨髓间充质干细胞(bone marrow derived mesenchymal stem cells,BMMSCs)外泌体(exosomes,Exo)对小胶质细胞(microglia,MG)M2型极化调控的影响。方法·分离、培养鼠BMMSCs,提取其Exo(BMMSCs-Exo),并分别对BMMSCs及BMMSCs-Exo行流式细胞术、透射电子显微镜(transmission electron microscope,TEM)与蛋白质印迹(Western blotting)检测及鉴定。合成miR-124-1基因,构建其慢病毒载体,观测过表达miR-124-1基因的BMMSCs及其Exo的miR-124-1基因的表达变化。将过表达miR-124-1基因的BMMSCs-Exo(BMMSCs-Exo+miR-124-1,Exo/124-1)与经脂多糖(lipopolysaccharide,LPS)活化的HAPI小胶质细胞株(HAPI细胞)共培养。分别收集Exo组与Exo/124-1组细胞。用仅含LPS培养基培养的HAPI细胞(LPS组)与未处理的HAPI细胞(正常组)作对照。使用实时荧光定量PCR(quantitative real-time PCR,qPCR)和Western blotting分别检测其M1型分子[白细胞介素-6(interleukin-6,IL-6)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)]和M2型分子(CD206、IL-10)的mRNA及其蛋白的表达变化。结果·成功分离、培养、鉴定了BMMSCs及BMMSCs-Exo。Exo/124-1明显表达miR-124-1基因。qPCR和Western blotting检测证实,BMMSCs-Exo能携带miR-124-1基因,其明显下调了HAPI细胞的IL-6(与正常组和LPS组比较,在基因水平分别下调了55.71%、73.04%,在蛋白水平分别为52.32%、76.95%)和TNF-α(与正常组和LPS组比较,在基因水平分别下调了51.95%、68.91%,在蛋白水平分别为52.14%、77.47%)。并且Exo/124-1上调了HAPI细胞的CD206(与正常组和LPS组比较,在基因水平分别上调了46.90%、76.99%,在蛋白水平分别为65.13%、85.11%)和IL-10(与正常组和LPS组比较,在基因水平分别上调了43.09%、72.36%,在蛋白水平分别为55.19%、78.18%)的表达。结论·BMMSCs-Exo可介导miR-124-1调控HAPI细胞向M2型极化。

Objective·To investigate the effect of exosomes(Exo)of bone marrow-derived mesenchymal stem cells(BMMSCs)overexpressing miR-124-1 gene on the regulation of transformation of M2 microglia(MG).Methods·BMMSCs of rats were isolated and cultured.BMMSCs-Exo were extracted.BMMSCs and BMMSCs-Exo were identified by flow cytometry,transmission electron microscope(TEM)and Western blotting,respectively.MiR-124-1 gene was synthesized and its lentiviral vector was constructed.The expression changes of miR-124-1 gene in BMMSCs and BMMSCs-Exo were observed.BMMSCs-Exo overexpressing miR-124-1 gene(Exo/124-1)was co-cultured with HAPI microglia cell line activated by lipopolysaccharide(LPS).Cells from Exo group and EXO/124-1 group were collected,respectively.HAPI cells cultured with LPS only(LPS group)were compared with untreated HAPI cells(normal group).Quantitative real-time PCR(qPCR)and Western blotting were used to detect the mRNA and protein expression of M1 molecules[interleukin-6(IL-6),tumor necrosis factor-α(TNF-α)]and M2 molecules(CD206,IL-10)in HAPI cells.Results·BMMSCs and BMMSCs-Exo were successfully isolated,cultured and identified.Exo/124-1 significantly expressed miR-124-1 gene.BMMSCs-Exo could carry miR-124-1 gene,significantly down-regulating the expression of IL-6(compared with the normal group and the LPS group,the down-regulation percentage were 55.71%and 73.04%,respectively at the gene level,and the percentages were 52.32%and 76.95%,respectively at the protein level)and TNF-α(compared with the normal group and the LPS group,the down-regulation percentage were 51.95%and 68.91%,respectively at the gene level,and 52.14%and 77.47%,respectively at the protein level)of HAPI cells tested by qPCR and Western blotting.Exo/124-1 up-regulated the expression of CD206(compared with the normal group and the LPS group,the down-regulation percentage were 46.90%and 76.99%,respectively at the gene level,and 65.13%and 85.11%,respectively at the protein level)and IL-10(compared with the normal group and the LPS group,the downregulation percentage were 43.09%and 72.36%,respectively at the gene level,and 55.19%and 78.18%,respectively at the protein level)in HAPI cells.Conclusion·BMMSCs-Exo can mediate miR-124-1 to regulate the polarization of HAPI cells to M2 type.