Objective To investigate the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the generation of human myeloid derived suppressor cells (MDSCs) relied on peripheral blood monocytes,and to establish efficient induction system in vitro of MDSCs.Methods Kidney transplantation recipients between January and March 2017 were included in this study.Purified CD14 + cells isolated from peripheral blood were cultured in the presence of GM-CSF with different concentrations for 7 days.Phenotypes and immunosuppressive abilities of induced MDSCs (iMDSCs) were investigated with FACS analyses.Inducible nitric oxide synthase (iNOS) mRNA expression was detected by qRT-PCR to determine the influence of iNOS-pathway on the immunosuppressive abilities of iMDSCs.Results A total of 11 recipients were included in this study.HLA-DR expression decreased sharply after the culture with GM-CSF.iMDSCs showed the similar phenotype characteristics with monocytic-MDSCs (M-MDSCs) as well as significant ability to suppress T cells proliferation and cytokines production.iMDSCs expressed higher levels of iNOS than monocytes,and the inhibitor effects of iMDSCs were significantly reduced after treatment with L-NMMA (1 mmol/L).The variations of phenotype and suppressive ability were concentrationdependent,and more significant changes could be revealed in the group of 10 μg/L GM-CSF.Conclusion GM-CSF-treated peripheral blood monocytes can be efficiently transformed to M-MDSCs,and suppress T cells proliferation and cytokines secretion via iNOS-dependent pathway.These results may contribute to establish MDSCs induction system,which would provide a basis for the clinical application of MDSCs.

Objective:To investigate the expression of microRNA (miR) -26a in human skin fibroblasts during photoaging induced by ultraviolet A (UVA) , and to evaluate the effect of up-or down-regulation of miR-26a expression on the methylation level of the whole genome, the target gene enhancer of zeste homolog 2 (EZH2) and cell aging.Methods:Some human skin fibroblasts were irradiated with 10 J/cm 2 UVA once a day for 7 consecutive days, RNA was extracted on days 0, 3 and 7, and real-time quantitative reverse PCR (RT-PCR) was performed to determine the expression of miR-26a; miR-26a mimics and inhibitors were transfected into fibroblasts to up-or down-regulate the expression of miR-26a respectively, and fluorescence microscopy and RT-PCR were performed to determine the expression of miR-26a and evaluate the transfection efficiency. Some human skin fibroblasts were divided into 6 groups: blank control group receiving no treatment, UVA group treated with UVA irradiation according to the above method, miR-26a mimic group transfected with miR-26a-mimics, UVA+miR-26a mimic group transfected with miR-26a-mimics followed by UVA irradiation, miR-26a inhibitor group transfected with miR-26a inhibitors, UVA+miR-26a inhibitor group transfected with miR-26a inhibitors followed by UVA irradiation. On day 7, cells in each group were collected after the end of UVA irradiation. Then, flow cytometry was performed to detect cell cycle, DNA methylation quantitative detection kit was used to detect the methylation level of whole genome, RT-PCR was conducted to determine the mRNA expression of EZH2 (a histone-lysine N-methyltransferase enzyme) , DNA methyltransferase 1 (DNMT1) and miR-26a, and Western blot analysis was performed to determine the protein expression of EZH2 and DNMT1. Statistical analysis was carried out by using one-way analysis of variance and least significant difference- t test. Results:Compared with the unirradiated control group, the expression of miR-26a gradually increased in the UVA irradiation group over time during the culture, and there was a significant difference in the expression of miR-26a between the two groups after 7 days of UVA irradiation ( t=5.295, P < 0.05) . Strong fluorescence signals were observed in the miR-26a mimic-or miR-26a inhibitor-transfected fibroblasts, suggesting a high transfection efficiency. Flow cytometry showed that the proportion of cells at G1 phase significantly differed among the blank control group, UVA group, miR-26a mimic group, UVA+miR-26a mimic group, miR-26a inhibitor group, and UVA+miR-26a inhibitor group (52.82% ± 2.56%, 78.56% ± 4.34%, 53.63% ± 3.13%, 89.52% ± 4.17%, 54.39% ± 3.86%, 65.34% ± 4.78%, respectively; F=46.728, P < 0.01) , and significantly higher in the UVA group than in the blank control group ( t=8.848, P < 0.01) , higher in the UVA+miR-26a mimic group than in the miR-26a mimic group and UVA group ( t=11.922, 3.154, P < 0.01, < 0.05, respectively) , and higher in the UVA+miR-26a inhibitor group than in the miR-26a-inhibitor group ( t=3.087, P < 0.05) , but significantly lower in the UVA+miR-26a inhibitor group than in the UVA group ( t=3.547, P < 0.05) . Detection of the genome-wide methylation level showed that the methylation level ( A450 value) significantly differed among the above groups (0.676 ± 0.024, 0.323 ± 0.043, 0.506 ± 0.035, 0.169 ± 0.024, 0.602 ± 0.036, 0.422 ± 0.029, respectively, F=97.402, P < 0.01) , and significantly lower in the UVA group than in the blank control group ( P < 0.01) , lower in the UVA+miR-26a mimic group than in the miR-26a mimic group and UVA group (both P < 0.01) , and lower in the UVA+miR-26a inhibitor group than in the miR-26a inhibitor group ( P < 0.01) , but significantly higher in the UVA+miR-26a inhibitor group than in the UVA group ( P < 0.05) . RT-PCR and Western blot analysis showed significant differences in the mRNA and protein expression of EZH2 and DNMT1 respectively among the 6 groups (both P < 0.05) , which were significantly lower in the UVA group than in the blank control group ( P < 0.05) , lower in the UVA+miR-26a mimic group than in the miR-26a mimic group and UVA group (both P < 0.05) , and lower in the UVA+miR-26a inhibitor group than in the miR-26a inhibitor group ( P < 0.05) , but significantly higher in the UVA+miR-26a inhibitor group than in the UVA group ( P < 0.05) . Conclusion:In the UVA irradiation-induced photoaging of skin fibroblasts, miR-26a expression was up-regulated, cellular proliferative activity and genome-wide methylation level decreased; up-regulation of miR-26a expression could down-regulate the expression of its target gene EZH2 and methylation-related gene DNM1, and promote cell photoaging, while down-regulation of miR-26a expression could up-regulate the expression of EZH2 and DNMT1, and inhibit cell photoaging.