The Research Of The Influence Of VIP On The Expression Of CD4 In Jurkat Cell Line And Its Possible Mechanism

Background:VIP?Vasoactive intestinal peptide,VIP?is a kind of common neuropeptide,which has been confirmed that can be secreted by tumor cells in different studies,and it has a regulatory effect on varieties of immune cells and can regulate the differentiation of helper cells.But the influence of VIP on the expression of CD4/CD8 in T lymphocyte has not been reported.Thpok is an important and necessary transcription factor during the T cell lineage differentiation,which promotes the double positive T cells into the CD4 single positive T cells during the positive selection,and maintains the expression of CD4 molecule but depresses the expression of CD8 molecule in mature T cells.The mutual antagonistic loop of Thpok and Runx3 is the keypoint in the CD4/CD8 lineage differentiation,and GATA3 showed a synergistic effect with Thpok.The differentiation process of T lymphocyte has been gradually clear,but few studies on the change of the phenotype of mature T lymphocyte have been reported so far.We found that the expression of CD4 in gastric cancer was significantly higher than that in the adjacent tissue while the transcription factor Thpok also showed the corresponding change in our previous study.Thus we hypothesized that tumor cells can induce the change of CD4/CD8 phenotype in mature T cells through secreting VIP,which may be related to Thpok and other transcription factors.So we designed the experiment in vitro to explore the influence of VIP on the expression of CD4 in jurkat cells and try to investigate its possible mechanism.Objective:To explore the influence of VIP on the expression of CD4 in jurkat cells and its possible mechanism.Methods:Firstly we detected the expression of CD4/CD8 and transcription factors Thpok,Runx3 and GATA3 in jurkat cells and molt-4 respectively.VIP was added to stimulate the na?ve jurkat cells and activated jurkat cells which was pretreated with PMA and ionomycin,flow cytometry was used to detect the changes of CD4 and CD8 expression of jurkat cells in each group post-stimulation.The m RNA and protein of Thpok?GATA3?Runx3 were detected by RQ-PCR and western-blot respectively.Finally we try to analyze the difference in CD4/CD8 expression between different lymphocyte cell lines jurkat and molt-4,and explore the possible mechanism of the difference of CD4 expression in each expremental group.Results:1.Both VPAC1 and VPAC2 were detected in jurkat cells.2.The expression of CD4 was significantly higher in jurkat cells,but the expression of CD8 was obviously higher in molt-4 cells?p<0.05?.The expression of Thpok and Runx3 in jurkat cells was higher than that in molt-4 cells?p<0.05?,but the difference of Thpok was much more notable.3.When stimulated with 10^-6M VIP,jurkat cells showed increasing expression of Thpok?p<0.05?,which indicated that 10^-6M was the concentration that in the experiments following.4.The expression of CD4 and CD8 raised in activated jurkat cells,and the increased tendency of CD4 can be enhanced when activated cells were costimulated with VIP?p<0.05?.5.a.The Thpok m RNA expression was up-regulated after jurkat cells were activated,and the tendency was enhanced when costimulated with VIP?p<0.05?.b.The expression of Runx3 m RNA and protein increased when jurkat cells were activated?p<0.05?.c.The expression of GATA3 protein was up-regulated when na?ve jurkat cells stimulated with VIP.Conclusions:1.The mature T lymphocyte jurkat still keeps plasticity,both CD4 and CD8 expression increased when jurkat cells were activated.2.VIP can up-regulate the expression of CD4 in activated jurkat cells,which may be related to Thpok.