人脐带间充质干细胞来源外泌体促进小胶质细胞向M2极化

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摘要目的: 探讨人脐带间充质干细胞外泌体（human umbilical cord mesenchymal stem cells exosomes, hucMSC-Ex）在小胶质细胞极化态转化中的作用及机制，为临床治疗神经退行性疾病提供新的治疗策略。方法: 原代分离培养人脐带间充质干细胞，收集其条件培养基提取外泌体（exosomes），电镜、纳米颗粒跟踪分析技术和蛋白质印迹法对其形态特征、粒径大小及表面标志物进行表征；分别用PBS、LPS（1 μg/mL）、LPS（1 μg/mL）+hucMSC-Ex（100 μg/mL）处理小胶质细胞12 h，免疫荧光检测小胶质细胞特异性标志物钙结合蛋白Iba1的表达；蛋白质印迹法检测小胶质细胞M1/M2极化态标志物变化；qRT-PCR检测小胶质细胞IL-1β mRNA和IL-10 mRNA的变化；ELISA检测细胞条件培养基中细胞因子IL-1β和IL-10的分泌量。结果： hucMSC-Ex电镜下呈现典型膜性“杯盘”状结构，纳米颗粒跟踪分析结果显示粒径大小为（119.7±47.9）nm，蛋白质印迹法证实其表达CD9、CD63和CD81特征表面分子；免疫荧光结果显示，活化组（LPS组和LPS+hucMSCEx组）钙结合蛋白Iba1相比于静息组（PBS组）表达高，且LPS+hucMSC-Ex组高于LPS组；蛋白质印迹法结合qRT-PCR结果显示hucMSC-Ex可能通过抑制NF-κB活化减少M1极化态标志物iNOS、IL-1β表达，上调M2极化态标志物Arg1、IL-10表达；ELISA结果表明相比于LPS组，LPS+hucMSC-Ex组分泌的炎症因子IL-1β减少、抑炎因子IL-10增加。结论： hucMSC-Ex可能通过抑制NFκB活化，促进小胶质细胞向M2极化而减少炎症反应。

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	周新茹
	金倩
	张蕾蕾
	吴佩佩
	傅强
	钱晖

关键词 ：间充质干细胞, 外泌体, 神经退行性疾病, 神经炎症, 小胶质细胞, 极化, NF-κB信号通路, 抑炎

Abstract：Objective: To investigate the role and mechanism of human umbilical cord mesenchymal stem cell-derived exosomes (hucMSC-Ex) in polarized transformation of microglia, and to provide a new treatment for clinical treatment of neurodegenerative diseases related to neuroinflammation. Methods: Primary human umbilical cord mesenchymal stem cells were isolated and cultured, and the conditioned medium was collected to extract exosomes. Electron microscopy, nanoparticle tracking analysis(NTA) technology and Western blotting technology were used to characterize their particle size, morphological characteristics and surface markers. Microglial cell lines were treated with PBS, LPS (1 μg/mL), LPS (1 μg/mL)+hucMSC-Ex (100 μg/mL) for 12 h, respectively.The expression of calcium-binding protein Iba1, a specific marker, was detected by immunofluorescence in different groups; Western blotting was used to detect changes of M1 and M2 microglia markers in different groups and to screen for possible signaling mechanisms; qRT-PCR was used to detect the changes of IL-1β mRNA and IL-10 mRNA in microglial cells in different groups; ELISA was used to detect the secretion of cytokines IL-1β and IL-10 in cell conditioned medium. Results: The electron microscope showed a typical membrane “cup and plate” structure, NTA results showed that the diameter of hucMSC-Ex was（119.7±47.9）nm and Western blotting confirmed that it expressed characteristic CD9, CD63, and CD81 surface molecules; immunofluorescence results showed that calcium-binding protein Iba1 in the activated group (LPS group, LPS + hucMSCEx group), was higher than the resting group (PBS group), and the LPS + hucMSC-Ex group was higher than the LPS group; Western blotting combined with qRTPCR results showed that hucMSC-Ex may reduce the expression of M1 microglia markers iNOS and IL-1β, and increase the expression of M2 microglia markers Arg1 and IL-10 by inhibiting NF-κB activation; ELISA results showed that compared with the LPS group, the inflammatory factor IL-1β decreased and the anti-inflammatory factor IL-10 increased in the LPS+hucMSC-Ex group. Conclusion: hucMSC-Ex may promote the polarization of microglia to M2 by inhibiting NF-κB activation and reduce the inflammatory response.

收稿日期: 2020-02-16

基金资助:国家自然科学基金资助项目(81971154)；镇江市外泌体基础与转化应用高技术研究实验室项目（SS2018003）

通讯作者: 钱晖（通讯作者），教授，博士生导师，E-mail：lstmmmlst@163.com；傅强（通讯作者），主任医师，副教授，博士生导师，Email：johson.f@163.com

作者简介: 周新茹(1995—), 女, 硕士研究生；

引用本文:

周新茹，金倩，张蕾蕾，吴佩佩，傅强，钱晖. 人脐带间充质干细胞来源外泌体促进小胶质细胞向M2极化[J]. 江苏大学学报:医学版, 2020, 20(04): 277-281. ZHOU Xin-ru1, JIN Qian1, ZHANG Lei-lei1, WU Pei-pei1, FU Qiang2, QIAN Hui1.

Human umbilical cord mesenchymal stem cell-derived exosomes promote microglial polarization to M2

. Journal of Jiangsu University(Medicine Edition), 2020, 20(04): 277-281.

链接本文:

http://zzs.ujs.edu.cn/xbyxb/CN/ 或 http://zzs.ujs.edu.cn/xbyxb/CN/Y2020/V20/I04/277

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