Regulatory effect of human umbilical cord mesenchymal stem cells-derived exosomes on macrophage polarization

doi:10.3969/j.issn.2095-4344.1685

Abstract

Abstract:

BACKGROUND: Accumulating evidence has suggested that mesenchymal stem cells-derived exosomes have the similar tissue repair activity compared to the mother cells. However, the underlying mechanisms are not yet illuminated. Since macrophages play pivotal roles during tissue repair process, we investigated the regulatory effect of mesenchymal stem cells-derived exosomes on macrophages, aiming to better elucidate its mechanism involved in tissue repair.
OBJECTIVE: To isolate exosomes from human umbilical cord-derived mesenchymal stem cells, and to evaluate their regulatory effect on macrophage polarization.
METHODS: Exosomes were isolated from the culture supernatants of human umbilical cord-derived mesenchymal stem cells by the differential centrifugation method, and then characterized by their morphology, size distribution and protein markers using transmission electron microscopy, nanoparticle tracking analysis and western blot assay, respectively. Furthermore, their regulatory effects on M1/M2 macrophage polarization were evaluated in mouse bone marrow-derived macrophages.
RESULTS AND CONCLUSION: Exosomes were successfully isolated from human umbilical cord-derived mesenchymal stem cell supernatants with a cup-shaped canonical morphology, (92.0±34.1) nm in diameter, and protein markers of CD63 and Alix. We determined that these exosomes could significantly reduce lipopolysaccharide-induced activation of M1 markers tumor necrosis factor-α and inducible nitric oxide, while upregulating M2 markers interleukin-10 andarginase-1. These findings suggest that exosomes from human umbilical cord-derived mesenchymal stem cells can regulate macrophage polarization to M2 phenotype, benefiting tissue repairing.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Umbilical Cord, Mesenchymal Stem Cells, Exosomes, Macrophages, Tissue Engineering

CLC Number:

Song Yuxian, Zhang Dongya, Xu Yujun, Hou Yayi, Ni Yanhong. Regulatory effect of human umbilical cord mesenchymal stem cells-derived exosomes on macrophage polarization[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(13): 2002-2008.

Figures/Tables 2

2.1 人脐带间充质干细胞的形态及表型鉴定用酶消化法获取人脐带间充质干细胞，在显微镜下观察细胞形态，可见细胞贴壁良好，呈现长梭形，见图1A；当细胞密度较大时呈涡漩状排列，见图1B。用流式细胞术检测细胞表面标志物，结果显示细胞阳性表达CD29、CD44、CD73、CD90和CD105，阴性表达CD11b、CD14、CD19、CD31和HLA-DR，见图1C，与文献报道的的间充质干细胞标准相符[20]。 2.2 外泌体的提取与鉴定收集人脐带间充质干细胞培养上清，采用经典的差速-超速离心方法提取外泌体[18]。采用Western blot检测外泌体常见标志物CD63和Alix的表达，其中CD63是一种四次跨膜蛋白，Alix是一种参与多泡体发生的蛋白，这两种蛋白在不同来源的外泌体中都有富集。结果显示CD63和Alix在人脐带间充质干细胞来源外泌体中有表达，而在人脐带间充质干细胞中则几乎检测不到，见图2A；透射电子显微镜观察外泌体形态，可见囊泡样结构，见图2B；粒径分析检测得知人脐带间充质干细胞来源外泌体的粒径为(92.0±34.1) nm，符合外泌体的基本特征[21-22]。 2.3 外泌体被巨噬细胞摄取用荧光标记物(PKH67-绿色)对外泌体进行标记后加入培养的巨噬细胞中，外泌体终质量浓度为10 mg/L，同时用脂多糖刺激，4 h后在荧光显微镜下可以看到大量外泌体被巨噬细胞摄取，见图3。 2.4 外泌体不影响巨噬细胞增殖活力和周期采用CCK-8实验检测细胞增殖活力，结果显示，10 mg/L外泌体对静息状态巨噬细胞、脂多糖活化状态巨噬细胞的增殖活力都没有影响，见图4A。细胞周期实验结果表明，该浓度的外泌体也不会影响细胞周期，见图4B，分别比较静息和活化状态下细胞周期G0/G1、S和G2/M期的百分比，发现外泌体处理组与不处理组差异均无显著性意义，见图4C。 2.5 外泌体促进巨噬细胞向M2型极化脂多糖刺激后巨噬细胞M1型活化标志肿瘤坏死因子α和一氧化氮合成酶的mRNA水平显著升高，而脂多糖+外泌体处理组与单独脂多糖组相比M1活化水平显著降低，见图5A，B；另外，M2型活化标志白细胞介素10和精氨酸酶1在脂多糖的作用下略有升高，而外泌体能显著上调M2的活化水平，见图5C，D。外泌体对M1和M2型活化标志蛋白水平的影响趋势与mRNA水平基本一致，即抑制M1而增强M2，见图6。"

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