Exosomes derived from human umbilical cord mesenchymal stem cells suppress Th22 expression and function

doi:10.3969/j.issn.2095-4344.0623

Abstract

Abstract:

BACKGROUND: Th22 cells are a novel type of CD4⁺ helper T cells. Exosomes derived from human umbilical cord mesenchymal stem cells (hUCMSCs) have T cell immunomodulatory function.
OBJECTIVE: To investigate the Th22 cell immunomodulatory ability of exosomes secreted from hUCMSCs．
METHODS: hUCMSCs were isolated and cultured, and cell line was authenticated using short tandem repeat (STR) analysis. Exosomes were extracted from the supernatant of passage 4 hUCMSCs. Morphology and particle size of the exosomes were detected under transmission electron microscope. The expression of specific surface marker CD63 and CD81 were detected by western blot. The concentration of exosomes was evaluated by BCA assay. Cell activation cocktail stimulated peripheral blood mononuclear cells from healthy donors were co-cultured with exosomes for 6 hours. The percentage of Th22 cells and the proliferation of CD3⁺CD4⁺T cells and CD3⁺CD8⁺T cells were detected by flow cytometry. Flow cytometry was also used to test the level of interleukin 22 at 72 hours of co-culture.
RESULTS AND CONCLUSION: Successfully established hUCMSCs primary cell line could stably passage. Exosomes secreted from hUCMSCs had typical exosomal morphology and marker proteins, and significantly inhibited the proliferation of CD3⁺CD4⁺T cells (P < 0.05) and CD3⁺CD8⁺T cells (P < 0.01). These exosomes also inhibited the proliferation of Th22 cells, and reduced the level of interleukin 22 in the cells (P < 0.01). These findings indicate that exosomes secreted from hUCMSCs inhibit Th22 cell expression and function, and have the immunomodulatory function of Th22 cells in vitro, which can be a new therapeutic agent for the treatment of immune disorders.

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, Immunomodulation, Tissue Engineering

CLC Number:

R394.2

Li Wei-wei, Li Xiao-feng, Hou Ping, Li Jian-ping. Exosomes derived from human umbilical cord mesenchymal stem cells suppress Th22 expression and function[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(29): 4657-4662.

Figures/Tables 2

2.1 hUCMSCs的生物学特性成功建立hUCMSCs细胞系，且可连续稳定传代(图1)。取稳定传代生长状态良好第8代细胞进行STR基因分型鉴定，确定此细胞系为课题组原代培养人源细胞系：与细胞株数据库上传细胞株基因数据无完全匹配，无错误鉴定和交叉污染(图2为鉴定报告结果部分截取图)。流式细胞检测发现，hUCMSCs稳定高表达CD90、CD73(>95%)，不表达CD45、HLA-DR(<2%)(图3)。 2.2 huc-exo鉴定结果应用细胞上清外泌体提取试剂盒，成功从第4代hUCMSCs中提取huc-exo。使用BCA法进行外泌体总蛋白浓度定量检测，测得样品蛋白质量浓度为(520±16.14) mg/L。透射电镜下可见大量直径在30-80 nm具备典型外泌体形态的囊性小颗粒(图4)。蛋白质免疫印迹法检测结果显示，huc-exo表达CD63、CD81蛋白(图5)。实验成功获取可供下游细胞培养实验所需要的较高浓度外泌体。 2.3 huc-exo对外周血单个核细胞体外增殖能力的影响经cell activation cocktail刺激后，外周血单个核细胞簇状集落样增殖明显。huc-exo组与阴性对照组相比较可抑制CD3+CD4+T细胞的增殖[(21.34±0.82)% vs.(16.13±0.45)%，P < 0.01，n=6，图6]；huc-exo组与阴性对照组相比较可显著抑制CD3+CD8+T细胞的增殖[(36.00±0.79)% vs. (19.76±0.29)%，P < 0.01，n=6，图6]。 2.4 huc-exo对Th22细胞体外增殖能力的影响经cell activation cocktail刺激后，外周血单个核细胞簇状集落样增殖明显。huc-exo组与阴性对照组相比较可显著抑制Th22细胞，即CD3+CD4+ IFN-γ-IL17-IL22+ T细胞的增殖[(0.71±0.16)% vs.(0.28± 0.07)%，P < 0.01，n=6，图7]。 2.5 huc-exo抑制Th22细胞分泌的细胞因子白细胞介素22含量经cell activation cocktail刺激后，外周血单个核细胞簇状集落样增殖明显。huc-exo组与阴性对照组相比较可显著抑制Th22细胞分泌的细胞因子白细胞介素22含量，与阴性对照组比较差异有显著性意义[(0.92±0.18)% vs. (0.48±0.07)%，P < 0.01，n=6，图8]。"

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