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

doi:10.3969/j.issn.1674-8115.2022.03.009

摘要/Abstract

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

中图分类号:

R392.2

郝磊, 金戈, 杨涌涛, 王军伟, 孙洋, 秦翠玲, 展群岭. 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[J]. 上海交通大学学报（医学版）, 2022, 42(3): 323-330.

HAO Lei, JIN Ge, YANG Yongtao, WANG Junwei, SUN Yang, QIN Cuiling, ZHAN Qunling. Effect of miR-124-1 mediated by exosomes of bone marrow-derived mesenchymal stem cells on the regulation of transformation of M2 microglia[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(3): 323-330.

图/表 9

表1 qPCR检测的引物序列

Tab 1 Primer sequences for qPCR

Gene	Forward primer （5'→3'）	Reverse primer （5'→3'）
TNF-α	ACCAGCAGATGGGCTGTACC	GCGGAGAGGAGGCTGACTTT
IL-6	CCACTGCCTTCCCTACTTCA	ACAGTGCATCATCGCTGTTC
IL-10	CGACGCTGTCATCGATTTCT	TAGATGCCGGGTGGTTCAAT
CD206	TGGGCTTATGGAGAGCCAAA	TCTGCAGTCACTGGTGGATT
Gapdh	TCAAGAAGGTGGTGAAGCAG	AGGTGGAAGAATGGGAGTTG

图1 镜下传代扩增的BMMSCsNote： Most of BMMSCs were spindle shaped and evenly distributed under inverted microscope.

Fig 1 BMMSCs subcultured and expanded under microscope

图2 流式细胞仪检测BMMSCs的CD44 (A)、CD90(B)与CD45(C)的阳性率

Fig 2 Detection of the positive rates of CD44 (A), CD90 (B) and CD45 (C) in BMMSCs by flow cytometry

图3 BMMSCs-Exo外泌体的鉴定Note： A. The shape of exosomes were oval and round vesicles under TEM. B. Detection of Exo marker proteins CD63 and CD9 by Western blotting. C. Quantitative analysis, ①P=0.000, vs the BMMSCs group.

Fig 3 Identification of BMMSCs-Exo

图4 重组慢病毒载体FT106/ miR-124-1的测序鉴定

Fig 4 Sequencing and identification of recombinant lentiviral vector FT106/miR-124-1

图5 重组病毒FT106/miR-124的包装Note： A. Under fluorescence microscope, normal 293T cells did not express GFP. B. 293T cells infected with FT106/miR-124-1 virus significantly expressed GFP.

Fig 5 Recombinant lentiviral vector FT106/miR-124-1 packaged with 293T cells

图6 BMMSCs及其Exo的重组病毒感染Note： A/B. Not infected by FT106/miR-124-1 virus (A) and infected by FT106/miR-124-1 virus (B) of BMMSCs. C. qPCR results. 1: Exo/124-1 group; 2: BMMSCs/124-1 group; 3: Exo group; 4: BMMSCs group. ①P=0.000, vs the Exo group; ②P=0.000, vs the BMMSCs group.

Fig 6 Recombinant lentivirus infection rate in BMMSCs and BMMSCs-Exo

图7 qPCR检测过表达miR-124-1的BMMSCs-Exo对HAPI细胞 CD206 （A）、 IL-10 （B）、 IL-6 （C）和 TNF-α （D）mRNA表达的影响Note： ①P=0.000, vs the LPS and the normal group.

Fig 7 Effects of overexpressing miR-124-1 gene in BMMSCs-Exo on the expressions of CD206, IL-10, IL-6 and TNF-α mRNA of HAPI cells detected by qPCR

图8 Western blotting检测过表达miR-124-1的BMMSCs-Exo对HAPI细胞CD206、IL-10、IL-6和TNF-α蛋白表达的影响Note： A. Western blotting results. B. Quantitative analysis results. ①P=0.000,vs the LPS and the normal group.

Fig 8 Effects of overexpressing miR-124-1 gene in BMMSCs-Exo on the expressions of CD206, IL-10, IL-6 and TNF-α protein in HAPI cells detected by Western blotting

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