| ObjectiveThe aim of this study was to research the effect of miRNA146 a on the differentiation and function of Th17 cells,then to explore the possible regulatory mechanisms.Eventually,to find a new way to regulate the immune response mediated by Th17 cells.MethodsMagnetic beads were used to separate the CD4+ CD25-naive T cells from mice spleen and the purity of the CD4+ CD25-naive T cells was confirmed by flowcytometry;the Dynabeads Mouse T-Activator CD3/CD28 was used to activate and proliferate the CD4+ CD25-naive T cells;then miR-146 a agomir and antagomir were used to transfect CD4+ CD25-naive T cells to regulate the expression level of miR-146 a,the experiment was divided into three groups,up-regulated group,down-regulated group and empty control group,and subgroups of different transfection concentrations were set in each group according to the preliminary results of research group for choosing the optimal transfection concentration;IL-6,TGF-?,anti-IL-4 antibody and anti-IFN-? antibody were used to induce the cells to differentiate from naive CD4+ CD25-T cells to Th17 cells and different induction schemes were used to induce cell differentiation to select the best induction scheme;detected the numbers of Th17 cells in each group by flowcytometry and the secretion of IL-17 in each group was detected by ELISA;QRT-PCR and Western blot were used to measure the expression levels of the possible target genes of miR-146 a.Results1.The percentage of CD4+ CD25-naive T cells in mouse spleen before and after sorting by magnetic beads were 24.9% and 92.3% respectively,and the cell activity was 98%.2.Successfully transfected and regulated the expression of miR-146 a in CD4+ CD25-naive T cells,and successfully induced CD4+CD25-naive T cells to differentiate into Th17 cells.3.The number of Th17 cells and the level of IL-17 secretion in supernatant cell culture were different in up-regulated groups,down-regulated groups and empty control groups after using the optimal concentration transfection of miR-146 a agomir or antagomir and the best induction scheme.Flowcytometry showed the number of Th17 cells in up-regulated groups was significantly increased compared with down-regulated groups and empty control groups(P<0.01);the number of Th17 cells in down-regulated groups was significantly decreased compared with empty control groups(P<0.01).The results of ELISA showed that the level of IL-17 secretion in up-regulated groups was significantly higher than down-regulated groups and empty control groups(P<0.01),the level of IL-17 secretion in down-regulated groups was significantly lower than that in empty control groups(P <0.01).4.QRT-PCR and Western blot showed the expression of the key transcription factor ROR-?t of Th17 cells and IL-17 gene were significantly higher in up-regulated groups than in other groups(P<0.01),the expression of STAT1 was insignificantly different in each group(P>0.05);the mRNA and protein expression levels of IRAK1 in up-regulated groups were significantly higher than in other groups,and the levels of it in down-regulated groups were significantly lower than in empty control groups(P<0.01).Conclusions1.The expression level of miR-146 a in CD4+ CD25-naive T cells was parallel with the differentiation of Th17 cells and the secretion of IL-17,up-regulated expression of miR-146 a could increase the differentiation number of Th17 cells and IL-17 secretion,while down-regulating the expression of miR-146 a,the number of Th17 cells and IL-17 secretion were decreased.2.Under the inflammatory environment that the Th17 cells are involved in,the expression level of STAT1 was insignificantly changed after regulating the expression level of miR-146 a,and in sustained inflammatory environment,miR-146 a was no longer negatively associated with another target gene-IRAK1.When the expression level of miR-146 a was up-regulated,the expression level of IRAK1 was also increased,which may contribute to the promotion of Th17 cell differentiation and the secretion of IL-17. |
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