脂多糖刺激的骨髓间充质干细胞来源外泌体改善小鼠心肌梗死后炎症和纤维化

doi:10.12092/j.issn.1009-2501.2019.08.001

中国临床药理学与治疗学 ›› 2019, Vol. 24 ›› Issue (8): 841-851.doi: 10.12092/j.issn.1009-2501.2019.08.001

• 基础研究 • 下一篇

脂多糖刺激的骨髓间充质干细胞来源外泌体改善小鼠心肌梗死后炎症和纤维化

傅小媚¹，霍然¹，邓赛¹，林潮金¹，范景皓¹，王萍¹，李松沛¹，秦爱萍¹，邹明辉^1,2，余细勇¹

¹广东省分子靶标与临床药理学重点实验室，广州医科大学药学院，广州 511436，广东；²分子与转化医学中心，乔治亚州立大学，乔治亚·亚特兰大 30314，美国

收稿日期:2019-03-07 修回日期:2019-05-24 出版日期:2019-08-26 发布日期:2019-08-30
通讯作者: 余细勇，男，博士，教授，博士生导师，研究方向：干细胞表观遗传药理学。 Tel：13724000603 E-mail：yuxycn@aliyun.com 邹明辉，男，博士，教授，博士生导师，研究方向：血管药理学。 E-mail：mzou@gsu.edu
作者简介:傅小媚，女，硕士，研究方向：心血管药理学。 Tel：15625127262 E-mail：15625127262@163.com
基金资助:
国家自然科学基金重点项目（U1601227）；广东省重大科技计划资助项目（2015B020225006）

Exosomes derived from LPS-stimulated bone marrow mesenchymal stem cell improve myocardial inflammation and fibrosis after myocardial infarction in mice

FU Xiaomei ¹, HUO Ran¹, DENG Sai ¹, LIN Chaojin ¹, FAN Jinghao ¹, WANG Ping ¹, LI Songpei¹, QIN Aiping¹, ZOU Minghui ^1,2, YU Xiyong¹

¹Guangdong Key Laboratory of Molecular Targets and Clinical Pharmacology, School of Pharmacy, Guangzhou Medical University, Guangzhou 511436, Guangdong, China; ² Center for Molecular and Translational Medicine, Georgia State University, Georgia, Atlanta 30314, USA

Received:2019-03-07 Revised:2019-05-24 Online:2019-08-26 Published:2019-08-30

摘要/Abstract

摘要：

目的: 探讨脂多糖（LPS）刺激下的骨髓间充质干细胞（BMSCs）来源的外泌体对小鼠心肌梗死（MI）后的治疗作用。方法: 流式成像细胞分析术鉴定BMSCs的表面标记分子，油红O和茜素红染色于镜下观察成骨成脂分化能力。LPS刺激BMSCs 48 h，提取细胞培养上清外泌体，用透射电镜和粒度分析仪捕捉其形态和粒径大小，流式细胞术和Western blot检测特异性表面标记分子及蛋白表达。建构小鼠MI模型并予以外泌体心脏局部注射，利用心脏超声、组织病理切片和PCR评价治疗结果。结果: 分离的BMSCs表达CD29、CD44、CD90、CD73、CD105，未表达HLA-DR、CD14、CD34、CD45，具有成骨成脂分化能力。Western blot检测到外泌体表达Alix、HSP70、Flotillin-1蛋白，流式成像仪检测到外泌体膜表面的CD63、CD9和CD81标记分子，粒径众数为69.2 nm。LPS刺激下BMSCs产生的外泌体注射小鼠心脏后，与PBS注射的MI对照组比较，心脏超声评价7天和28天的射血分数（EF）和短轴缩短率（FS）都显著增大（P<0.01,P<0.05），在第28天的舒张末期容积（LVEDV）和收缩末期容积（LVESV）均显著减小（P<0.05），Masson染色显示纤维化面积减小（P<0.01），PCR检测到炎症因子IL-6、IL-1β、转化生长因子β（TGF-β）和趋附因子CCL2表达减少，CX3CL1表达增多，术后第14天最为明显。结论: LPS刺激的BMSCs来源外泌体可以改善小鼠MI后心肌收缩功能和纤维化，降低炎症因子表达量。

关键词: 骨髓间充质干细胞, 外泌体, 脂多糖, 心肌梗死, 纤维化, 炎症

Abstract:

AIM: To investigate the therapeutic effect of exosome derived from lipopolysaccharide (LPS)-stimulated bone marrow mesenchymal stem cells (BMSCs) on myocardial infarction (MI) in mice. METHODS: Flow cytometry was used to identify the surface marker of BMSCs. Oil red O and alizarin red staining were used to observe the differentiation to osteoblasts and Lipid. Exosomes were extracted from supernatant of BMSCs stimulated by LPS for 48 h, captured its morphology and particle size by transmission electron microscopy and particle size analyzer. Flow cytometry and Western blot were used to detect specific surface marker and protein expression. The mouse MI model was constructed and the exosomes were local injected into the heart, the therapeutic effects were evaluated by echocardiography, histopathological sections and PCR. RESULTS:Isolated BMSCs expressed CD29, CD44, CD90, CD73 and CD105, but not HLA-DR, CD14, CD34, CD45, and BMSCs had osteogenic and adipogenic differentiation ability. The expression of protein Alix, HSP70 and Flotillin-1 in exosomes was detected by Western blot. The surface markers CD63, CD9 and CD81 of the exosomes were detected by flow cytometry. The mode of the particle size was 69.2 nm. After heart injection of exosomes derived from LPS-stimulated BMSCs, compared with the PBS-injected MI control group, the EF and FS scores on day 7 and day 28 calculated by echocardiography were significantly increased (P<0.01, P<0.05), and the LVEDV and LVESV on day 28 were significantly increased (P<0.05). Masson staining showed fibrosis area decreased (P<0.01), The expression of inflammatory factors IL-6, IL-1β, TGFβ and CCL2 detected by PCR was decreased, and the expression of CX3CL was increased, which was the most obvious on the 14th day after operation.CONCLUSION: LPS-stimulated BMSC-derived exosomes can improve myocardial contractile function and fibrosis after myocardial infarction in mice, and reduce the expression of inflammatory factors.

Key words: bone marrow mesenchymal stem cells, exosomes, lipopolysaccharide, myocardial infarction, fibrosis, inflammation

中图分类号:

傅小媚，霍然，邓赛，林潮金，范景皓，王萍，李松沛，秦爱萍，邹明辉，余细勇. 脂多糖刺激的骨髓间充质干细胞来源外泌体改善小鼠心肌梗死后炎症和纤维化[J]. 中国临床药理学与治疗学, 2019, 24(8): 841-851.

FU Xiaomei, HUO Ran, DENG Sai, LIN Chaojin, FAN Jinghao, WANG Ping, LI Songpei, QIN Aiping, ZOU Minghui, YU Xiyong. Exosomes derived from LPS-stimulated bone marrow mesenchymal stem cell improve myocardial inflammation and fibrosis after myocardial infarction in mice[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2019, 24(8): 841-851.

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脂多糖刺激的骨髓间充质干细胞来源外泌体改善小鼠心肌梗死后炎症和纤维化

Exosomes derived from LPS-stimulated bone marrow mesenchymal stem cell improve myocardial inflammation and fibrosis after myocardial infarction in mice

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