IL-17对人脐带间充质干细胞体外免疫调节特性的影响

Effect of IL-17 on the Immunoregulatory Characteristics of Human Umbilical Cord Mesenchymal Stem Cells in Vitro

目的:研究白细胞介素17(IL-17)对人脐带间充质干细胞(hUC-MSCs)的免疫调节能力的影响。方法:采用组织块贴壁法从健康足月胎儿脐带中分离培养hUC-MSCs,并对其进行鉴定。然后在培养基中加入不同浓度IL-17,采用EdU细胞增殖法检测hUC-MSCs增殖情况,流式细胞术(FCM)检测h UC-MSCs表面标记及细胞周期,RT-qPCR检测免疫调节相关基因表达水平,ELISA检测上清中免疫调节相关因子分泌水平,免疫学反应检测其免疫调节能力。结果:①倒置显微镜下观察可见hUC-MSCs呈现较均一漩涡状、梭形生长,细胞体积较小;细胞表面表达CD44、CD73、CD90、CD105,不表达CD34、CD19、CD11b、CD45、HLA-DR;具有成脂、成骨、成软骨诱导分化能力。②FCM检测结果显示IL-17处理后,hUC-MSCs免疫表型未发生变化,而IFN-γ处理后,hUC-MSCs表面标记HLA-DR表达明显上调。③与对照组相比,IL-17处理组的hUC-MSCs活性更好,且能促进细胞增殖。④与空白对照组相比,IL-17处理组h UC-MSCs的TGF-β、IL-10、IDO、PGE2、TSG-6、PD-L1基因表达均显著上调(P<0.001)。⑤ELISA检测结果显示,与空白对照组相比,IL-17处理组上清中TGF-β、IL-10、IDO、PGE-2、TSG-6、PD-L1分泌水平明显增高(P<0.001)。⑥与空白对照组相比,IL-17处理组的CD3+CD4+T细胞、CD3+CD8+T细胞、B细胞的比例降低,Treg细胞比例增高(P<0.001)。结论:IL-17在不影响hUC-MSCs免疫表型的条件下,可促进细胞增殖,上调免疫相关基因表达,提高TGF-β、IL-10、PGE-2、IDO、TSG-6、PD-L1分泌水平,增强hUC-MSC免疫调节能力。因此,本研究将为预处理的hUC-MSCs防治免疫炎症性疾病提供理论依据。

Objective: To observe the effects of interleukin 17(IL-17) on the immunomodulatory capacity of human umbilical cord mesenchymal stem cells(h UC-MSCs). Methods: h UC-MSCs were isolated from the umbilical cord of healthy term fetus by tissue explants adherent method, and cultured with serum-free medium with IL-17. EdU cell proliferation method was used to detect the proliferation of h UC-MSCs, Flow cytometry was used to detect cell surface markers and cell cycle, gene expression levels related to immune regulation was detected by RT-q PCR, the secretion level of immunomodulatory factors was detected by ELISA, and the immunomodulatory ability of hUC-MSCs was detected by immunological experiment. Results: ① Under the inverted microscope,hUC-MSCs cultured with with serum-free medium showed more uniform vortex-like and spindle growth. hUC-MSCs were expressed CD44, CD73, CD90 and CD105, but lowly expressed CD34, CD19, CD11 b, CD45, and HLA-DR. Results from the induced differentiation experiments showed that h UC-MSCs had adipogenic, osteogenic and chondrogenic abilities. ②After IL-17 pre-treatment,the immunophenotype of hUC-MSCs was not changed, however, after IFN-γ pre-treatment, the expression of HLA-DR on hUC-MSCs was significantly up-regulated. ③ As compared with control group, the cell viability and cell proliferation of hUC-MSCs were pre-treatment by IL-17 was better. ④Compared with the blank control group, the gene expressions of TGF-β, IL-10, IDO, PGE2, TSG-6,and PD-L1 of hUC-MSCs were pre-treatment by IL-17 were significantly up-regulated(P<0.001). ⑤Compared with the blank control group, the secretion levels of TGF-β, IL-10, IDO, PGE-2, TSG-6 and PD-L1 were significantly increased in the supernatant of UC-MSCs were pre-treatment by IL-17 group(P<0.001). ⑥hUC-MSCs were pre-treatment by IL-17 can reduce the proportion of CD3+CD4+T cells,CD3+CD8+Tcells, and B cells in lymphocytes, but increase the proportion of Treg cells(P<0.001). Conclusions: Without affecting the immunophenotype of hUC-MSCs, IL-17 can promote cell proliferation, up-regulate the expression of immune-related genes, increase the secretion level of TGF-β, IL-10, PGE-2, IDO, TSG-6, PD-L1, and enhance immune regulation ability of hUC-MSC. Therefore, this study will provide a theoretical basis for priming h UC-MSCs to prevent and treat immune inflammatory diseases.