| Objective To explore whether tumor necrosis factor receptor GITR can be used as a better surface marker than CD25 for the identification of Treg,and to try to establish a method for in vitro amplification of CD4~+Treg.Methods BALB/c and C57BL/6 mice at 2~-3 weeks,8~-10 weeks and 40~-48 weeks of age were selected.The thymus and spleen cells labeled with CD3,CD4,CD25 and GITR were analyzed by flow cytometry.CD3~+CD4~+CD25~+,CD3~+CD4~+GITR~+,CD3~+CD4~+GITR~-,and CD3~+CD4~+CD25~+GITR~+cells were sorted by flow cytometry and analyzed by Fox P3 staining.CD4~+CD25~-cells from spleen cells of C57BL/6 mice were selected by flow cytometry as the experimental group(unselected spleen cells as the control group),stimulated by anti~-CD3 /CD28,and induced by TGF~-β and IL~-2 at the same time.After 4days of co~-culture,the proportion of CD4~+CD25~+Treg cells in cultured cells was detected by flow cytometry.CD3~+CD4~+CD25~+,CD3~+CD4~+GITR~+,CD3~+CD4~+GITR~-and CD3~+CD4~+CD25~+GITR~+ cells were sorted by flow cytometry and analyzed by Fox P3 staining.Meanwhile,the expression of Foxp3 protein in C57BL/6 mouse spleen cells was detected by Western blot and compared with that in C57BL/6 mouse spleen cells(blank group).The m RNA expression levels of Foxp3 in the three groups were detected by RT~-PCR.Results FCM results showed that the proportion of CD4~+CD25~+ and CD4~+GITR~+cells in T lymphocytes of thymus and spleen of BALB/c and C57BL/6 mice aged2~-3 weeks,8~-10 weeks and 40~-48 weeks was similar,and the proportion of each cell group in adult mice was higher.Compared with the other two groups,the proportion of the aged group decreased(P<0.05).Although there was a slight difference in the cell proportion between BALB/C and C57BL/6 mice,the expression of the two molecules in the spleen of adult C57BL/6 mice was more similar and stable.Fox P3 staining showed collective migration of the two groups of cells,and loss of CD25 and GITR staining signals.FCM detected the results of Fox P3 intracellular staining in CD4~+CD25~-cells(experimental group),whole spleen cells(control group)and spleen cells of C57BL/6 mice without any induction treatment(blank group)by flow cytometry.Experimental group > control group > blank group,and the experimental group and control group were significantly different from blank group(P<0.05,P<0.01).The positive rate of CD3~+CD4~+CD25~+ and CD3~+CD4~+GITR~+ cells in the experimental group was over 90%,which was much higher than that in the control group.When CD3~+CD4~+CD25~+,CD3~+CD4~+GITR~+,CD3~+CD4~+GITR~-,and CD3~+CD4~+CD25~+GITR~+ cells were sorted by flow cytometry,the number of cells obtained from other groups was very small except CD4~+CD25~+GITR~+.Western blot was used to detect the expression of Foxp3 protein in CD3~+CD4~+CD25~+GITR~+ group in experimental and control cells,and the natural blank group was used as a reference for quality control.The results showed as follows: Experimental group > control group > blank group,Fox P3 protein expression was increased in each group compared with blank group,the difference was statistically significant(P<0.05,P<0.01).RT~-PCR detection of Foxp3 m RNA expression showed that the experimental group > control group >blank group,there was no significant difference between the control group and the experimental group,but the control group and the experimental group were significantly different from the blank group(P<0.05,P<0.01).Conclusions The expression of tumor necrosis factor receptor GITR induced by glucocorticoid is highly consistent with CD25,which can be used as one of the surface markers of Treg.In vitro induction of CD4~+CD25~-cells with anti~-CD3/CD28 stimulation and TGF~-βand IL~-2 at the same time can obtain higher effective proliferation of Treg than that of whole spleen cells in vitro induction,and its function and stability need to be further studied. |
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