The Mechanism Of Tn Antigen Expression In T Cells Induced By Activator In Vitro

1.The mechanism of Tn antigen expression in T cells stimulated by CD3/CD28 in vitroObjective:To explore the expression of Tn antigen on T cells stimulated by CD3/CD28.Methods:30 cases of healthy adult anticoagulation blood were selected from Yantai central blood station.Ficoll density gradient centrifugation was used to isolate PBMC.The CD3+T cells in PBMCs were purified by negative selection of the MACS isolation and incubated in 37? cell incubator.Then,CD3+T cells were stimulated with CD3/CD28 for 12h,24h,36h,48h,60h,72h,respectively.Flow cytometry was used to detect the percentage of Tn antigen expression on CD4+,CD8+T cells in different time periods before and after activation.The mean fluorescence intensity of Tn antigen on T cells was analyzed statistically.The Cosmc gene was amplified by qPCR and RT-PCR.Western-blot was used to detect the expression of Cosmc protein in T cells at each activation time.The level of Cosmc protein were detected by Western blot.Fluorescent assay was used to detect the activity of T-synthase in T cells at each time points.ELISA assay was used to detect the secretion of L-4 and IFN-y in the culture supernatant.BSP method was used to detect the methylation status of Cosmc promoter before and after activation.Results:Tn antigen is expressed in activated CD4+T cells and that the percentage of positive(Tn+)cells increased first and then decreased within 72h after stimulation.The Tn antigen expression peaked at 48h after stimulation with T cell activator CD3/CD28.During activation,the change in IL-4 levels of T cell culture supernatant was consistent with the change in percentage of Tn+ cells,whereas reverse trend was observed IFN-?secretion.T-synthase activity and Cosmc transcription decreased,accompanied by further increase in methylation of Cosmc promoter.The Cosmc gene and transcription level also appeared to decrease first and then increase,and Cosmc promoter methylation is dynamic processes during T cell activation.Conclusion:High methylation of Cosmc promoter and SP1/SP3-binding pionts may result in the expression of Tn antigen in activated T cells.2.Study on the relationship of Tn antigen and Cosmc in T cells stimulated by Cell Stimulation CocktailObjective:To investigate the relationship between the expression of Tn antigen and Cosmc of T cells activated by Cell Stimulation Cocktail in vitro.Methods:Ficoll density gradient centrifugation was used to isolate healthy human peripheral blood mononuclear cells(PBMC).The CD3+T cells in PBMCs were purified by negative selection of the MACS isolation and incubated in 37 ? cell incubator.Then,CD3+T cells were stimulated with Cell Stimulation Cocktail for 12h,24h,36h,48h,60h,72h,respectively.Flow cytometry(FCM)was used to detect the percentage of Tn+ cells and the mean fluorescence intensity of Tn antigen in T cells at different time points before and after activation.The Cosmc gene was amplified by qPCR and RT-PCR.Western-blot was used to detect the expression of Cosmc protein in T cells at each activation time.Fluorescent assay was used to detect the activity of T-synthase in T cells at each time points.Results:Cocktail could lead to Tn antigen expression on the T cells surface.The percentage of Tn+ cells increased first and then decreased along with time.The levels of Cosmc transcription,protein and the T-synthase activity of activated T cells showed a tendency to decrease first and then increase.This change was opposite to that of Tn antigen.Conclusion:Tn antigen expression was dynamically altered during T cells stimulated by Cocktail in vitro.The expression level of Tn antigen is closely related to the decrease of Cosmc transcription,protein level and T-synthase activity.