Activation Analysis of C57BL/6 Mouse Bone Marrow Derived Dendritic Cells Stimulated by Fusion Protein CTP-FoxM1

Abstract: The tumour associated antigens of DCs (dendritic cells) can significantly increase the expression of cell surface molecules MHC-II and costimulatory molecules such as CD40， CD80 and CD86， enhance the Th1 type cytokines secretion and present the antigens to T cells to induce the anti-tumour immune response. Whether the tumour associated antigens can fully activate DCs is the key factor during the whole anti-tumour immune response. In order to detect the effect of the fusion protein CTP-FoxM1 on the activation of C57BL/6 mouse bone marrow derived DCs， CTP-FoxM1 fragments were sequentially inserted into plasmid pCold-TF， then the constructed recombinant plasmid was transformed into E.coli BL21 and induced by IPTG. The fusion protein CTP-FoxM1 was purified by affinity chromatography and finally identified by Western blot assay. The survival rates of DCs treated with different concentrations of CTP-FoxM1 for 48 h were tested by the CCK-8 assay; the cytoplasmic localization of the fusion protein was observed by the confocal microscopy; the expressions of MHC-II， CD40， CD80 and CD86 phynotypes of DCs were evaluated by the flow cytometry and the secretion of cytokine IL-12 was detected by the ELISA assay. The result indicated that the survival rates were surpressed by the fusion protein CTP-FoxM1 in a concentration depended manner. The survival rate of 1 μg/mL of CTP-FoxM1 treated DCs (80.93%±8.36%) was higher than that of the 2 μg/mL of CTP-FoxM1 treated DCs (70.13%±5.38%， P<0.001) and 4 μg/mL of CTPFoxM1 treated DCs (62.97%±4.06%， P<0.001). The fusion protein CTP-FoxM1 was located in the cytoplasm of DCs. It could upregulate the expression levels of MHC-II， CD40， CD80 and CD86 of DCs and the secretion of IL-12 (P<0.001) compared with PBS treated groups. The study suggests that the fusion protein CTP-FoxM1 has the capacity of activating DCs and the potency of immunotherapy of hepatocellular carcinoma. These findings make a foundation for the further study of the immunological effects of DCs pulesd with CTP-FoxM1 in vivo.

CSTR: 32200.14.cjcb.2015.05.0006