The Preliminary Study About The Immune Activation Effect Of Fusion Protein CTP-FOXM1 On The C57BL/6 Mouse Bone Marrow Derived Dendritic Cell

Objective:To detect the immune activation effect of the fusion protein CTP-FoxM1 on the C57/BL6 mouse bone marrow derived DCs, making further study for its immunological effect in vivo.Methods:(1) Bone marrow cells were obtained in vivo under sterile conditions, Erythrocytes were lysied, the cells were cultured in RPMI1640 media containing GM-CSF and IL-4 for 7d. The morphage of DCs was observed under macroscope, the phynotypes of DCs were identified through flow cytometry, the IL-12 releasing level was tesed by ELISA, the ability of stimulating T cells were detected through MLR test.(2) DCs were treated with the fusion protein CTP-FoxM1、 protein CTP and protein FoxM1 for 48h, the survival rates were tested by CCK-8 assay, the phenotypes of DCs treated with the three kinds of proteins were tested by flow cytometry, the releasing quantity of IL-12 were measured by ELISA.(3) The DCs were treated with CTP-FoxM1、CTP、FoxM1 for 48h and then cocultured with CD8+T cells for 72h, the releasing level of IFN-y and TNF-a were tested by ELISA.Results:(1) After the mouse bone marrow derived cells were obtained under sterile condition and induced by GM-CSF and IL-4 for 7d, we could gain 3×107cells per mouse, the expression of characteristic phenotype CD11c was about 88±4.66%, MLR testes showed DCs has the ability of stimulating the proliferation of T cells(P<0.01). Compared with immature DCs, the DCs treated with LPS showed high expressions of surface molecules and high level of IL-12 releasing(P<0.001).(2) The growth of DCs can be surpressed by the fusion protein CTP-FoxM1 in a concentration depended manner, Compared with CTP and FoxMl treated groups, CTP-FoxM1 treated groups showed significantly high level of expression of surface molecules and IL-12 releasing quantity.(2) The releasing level of IFN-y and TNF-a were higher than the CTP treated groups (P<0.01) and FoxM ltreated groups after cocultrued with CD8+T cells for 72h(P<0.001).Conclusion:(1) High purity DCs can be gained from our modified cultured method for the further study.(2) Fusion protein CTP-FoxMl has the potential for immune activation of DCs by upregulating the expression of DCs molecules and IL-12 releasing level.(3) DCs treated with CTP-FoxMl can upregulate the expression of cytokine IFN-γ、TNF-α after cocultured with CD8+T cells, suggesting it can induce DCs to develop the antigenic cross-activity and make CD8+Tcells to play an important role in the anti-tumour immune response.