上海交通大学学报(医学版) ›› 2022, Vol. 42 ›› Issue (3): 323-330.doi: 10.3969/j.issn.1674-8115.2022.03.009

• 论著 · 基础研究 • 上一篇    下一篇

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

郝磊(), 金戈, 杨涌涛, 王军伟, 孙洋, 秦翠玲, 展群岭()   

  1. 重庆市第五人民医院神经内科,重庆 400062
  • 收稿日期:2022-01-04 出版日期:2022-03-28 发布日期:2022-05-09
  • 通讯作者: 展群岭 E-mail:haolei1102@163.com;aczhan@163.com
  • 作者简介:郝磊(1982—),女,副主任医师,博士;电子信箱:haolei1102@163.com
  • 基金资助:
    重庆市自然科学基金(cstc2020jcyj-msxmX0988)

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

Lei HAO(), Ge JIN, Yongtao YANG, Junwei WANG, Yang SUN, Cuiling QIN, Qunling ZHAN()   

  1. Department of Neurology, the Fifth People's Hospital of Chongqing, Chongqing 400062, China
  • Received:2022-01-04 Online:2022-03-28 Published:2022-05-09
  • Contact: Qunling ZHAN E-mail:haolei1102@163.com;aczhan@163.com
  • Supported by:
    Natural Science Foundation of Chongqing(cstc2020jcyj-msxmX0988)

摘要:

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。

关键词: 骨髓间充质干细胞, 外泌体, miR-124-1基因, HAPI细胞, M2型极化

Abstract:

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 down-regulation 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.

Key words: bone marrow-derived mesenchymal stem cell (BMMSC), exosome (Exo), miR-124-1 gene, HAPI cell, the polarization of M2 type

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