| 摘要: |
| ［摘要］ 目的 探讨miR-155调控巨噬细胞极化并经外泌体介导足细胞损伤的作用机制。方法 选择巨噬细胞RAW264.7和肾小球足细胞MPC5进行实验。将miR-155 mimics、miR-155 mimics NC、miR-155 inhibitor和miR-155 inhibitor NC慢病毒转染至RAW264.7细胞，构建miR-155过表达/低表达巨噬细胞系及其对照，分离巨噬细胞外泌体。采用流式细胞术检测各组巨噬细胞M1/M2表型比值。采用Transwell法将RAW264.7细胞与MPC5细胞进行共培养，分别于共培养12 h、24 h后采用实时荧光定量聚合酶链反应（RT-qPCR）检测巨噬细胞miR-155表达水平；采用Western blot法检测巨噬细胞IL-6、IL-10表达水平，以及肾小球足细胞synaptoporin和nephrin表达水平；采用酶联免疫吸附试验（ELISA）检测肾小球足细胞IL-1β、IL-10表达水平；采用TUNEL染色法检测肾小球足细胞凋亡情况。结果 成功构建miR-155过表达/低表达巨噬细胞系并分离其外泌体。miR-155过表达可诱导巨噬细胞极化为M1型，抑制miR-155可诱导巨噬细胞极化为M2型。在miR-155 mimics慢病毒转染至巨噬细胞24 h后，其miR-155表达水平显著增加（P<0.05），并上调IL-6的表达水平（P<0.05），抑制IL-10的表达水平（P<0.05），经Transwell共培养使足细胞synaptoporin、nephrin表达水平下降（P<0.05），IL-1β表达水平升高（P<0.05），并促进足细胞发生凋亡。而转染miR-155 inhibitor慢病毒至巨噬细胞后所得结果趋势与miR-155 mimics相反。RT-qPCR检测结果显示，miR-155 mimics组巨噬细胞外泌体中miR-155水平显著升高（P<0.05），miR-155 inhibitor组巨噬细胞外泌体中miR-155水平显著降低（P<0.05）。结论 miR-155过表达可诱导巨噬细胞极化为M1型，并通过外泌体介导引发足细胞损伤，而下调miR-155表达则逆转这一作用。 |
| 关键词: miR-155 巨噬细胞 外泌体 足细胞损伤 凋亡 炎症因子 |
| DOI：10.3969/j.issn.1674-3806.2024.08.03 |
| 分类号: |
| 基金项目:国家自然科学基金项目（编号：82060133，81860131）；广西自然科学基金项目（编号：2017GXNSFAA198288，2019GXNSFDA245004） |
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Study on the mechanism of action of miR-155 regulating the polarization of macrophages and mediating
podocyte injury through exosomes |
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XU Luyao1,2, ZHANG Jie1,2, LIN Xu2,3
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1.Graduate School, Youjiang Medical University for Nationalities, Baise 533000, China; 2.Department of Nephrology, Affiliated Hospital of Youjiang Medical University for Nationalities, Baise 533000, China; 3. Guangxi Key Laboratory of Basic Medical Research Support for Immune-related Diseases, Baise 533000, China
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| Abstract: |
| ［Abstract］ Objective To explore the mechanism of action of micro ribonucleic acid-155(miR-155) regulating the polarization of macrophages and mediating podocyte injury through exosomes. Methods Macrophage RAW264.7 and glomerular podocyte MPC5 were selected for the experiments. The miR-155 mimics, miR-155 mimics negative control(NC), miR-155 inhibitor and miR-155 inhibitor NC lentiviruses were transfected into RAW264.7 cells, and miR-155 overexpressing/underexpressing macrophage cell lines and their controls were constructed and the macrophage exosomes were isolated. Macrophage M1/M2 phenotype ratio in each group was detected by using flow cytometry. RAW264.7 cells were co-cultured with MPC5 cells by using Transwell method, and the miR-155 expression level of macrophages was detected by using real-time fluorescence quantitative polymerase chain reaction(RT-qPCR) after 12 hours and 24 hours of co-culture, respectively, and the expression levels of interleukin(IL)-6 and IL-10 in macrophages, as well as the expression levels of synaptoporin and nephrin in glomerular podocytes were detected by using Western blot. The expression levels of IL-1β and IL-10 in glomerular podocytes were detected by using enzyme-linked immunosorbent assay(ELISA), and the apoptosis of glomerular podocytes was detected by using TUNEL staining method. Results The miR-155 overexpressing/underexpressing macrophage cell lines were successfully constructed and their exosomes were successfully isolated. Overexpression of miR-155 could induce the polarization of macrophages to M1 type, and inhibition of miR-155 could induce the polarization of macrophages to M2 type. After miR-155 mimics lentiviruses were transfected into macrophages for 24 hours, their miR-155 expression levels were significantly increased(P<0.05), and the expression levels of IL-6 were up-regulated(P<0.05), and the expression levels of IL-10 were inhibited(P<0.05), and the co-culture by using Transwell resulted in decrease in the expression levels of synaptoprin and nephrin in podocytes(P<0.05), and increase in the expression level of IL-1β and facilitated podocyte apoptosis. However, the trend of the results obtained after transfection of miR-155 inhibitor lentiviruses into macrophages was opposite to that of miR-155 mimics. The results of RT-qPCR detection showed that the level of miR-155 in macrophage exosomes was significantly increased in the miR-155 mimics group(P<0.05), and the level of miR-155 in macrophage exosomes in the miR-155 inhibitor group was significantly decreased(P<0.05). Conclusion Overexpression of miR-155 can induce macrophages to polarize into M1 type and induce podocyte injury mediated by exosomes, while downregulation of miR-155 expression reverses this effect. |
| Key words: Micro ribonucleic acid-155(miR-155) Macrophage Exosome Podocyte injury Apoptosis Inflammatory factor |
