The Study Of Biological Characteristics Of Dendritic Cells Vaccines Transfected By IL-15 Gene And Loaded With Tumor Antigens

Objective: To study the biological characteristics of dendritic cells vaccines transfected by IL-15 gene and loaded with tumor antigens.Methods: 1.First,the mIL-15 cNDA were obtained from the mouse renal cNDA by pfxDNA polymerase. Next, mIL-15 cNDA was cloned into pGEM-T Easy Vector,obtained the mIL15-T cloning vehicle.Third,mIL-15 cNDA was cloned into adenovirus transfer vector pShuttle-GFP-CMV ofter digestion from the mIL-15-T cloning vehicle by EcoRI. So we obtained the pShuttle-GFP-mIL-15 plasmid, then pShuttle-GFP-mIL-15 was transferred into pAdxsi vector to construct Ad-GFP-mIL-15 plasmid. The correct adenoviral recombinant was produce and purify.2.The precursor dendritic cells was obtained from the C57BL/6 mouse bone marrow. Then the precursor dendritic cells were developed under the concentration of rmGM-CSF,rmIL-4 in vitro. Observing the morphology of the precursor dendritic cells, The phenotypic characterization of the immaturity dendritic cells were analyzed with flowcytometry. Collection the immaturity dendritic cells in 5-days, transfecting recombinant adenovirus expression plasmid IL-15 and inducing tumor lysates into the immaturity dendritic cells. It was the full dendritic cells vaccines transfected by IL-15 gene and loaded with tumor antigens (IL-15/lysate-DC) now. The capacity of stimulating T cells was determined by allogeneic IL-15/lysate-DC vaccines, The capacity of killing tumor cells was determined by cytotoxic T lymphocyte reaction(CTL).Results: 1.As confirmed by restriction digestion analysis, electrphoresis, sequencing and RT-PCR, we successfully constracted the recombinant adenovirus vector encoding IL-15 gene, and it have the IL-15 gene order in the insertion element correctly. 2. Flowcytometry analysis dected high expression of the costimulation molecules CD80, CD86, CD40,CD11c and MHC-â…¡i n IL-15/lysate-DC.The CD80,CD40 of IL-15/lysate-DC vaccines was higher than the other DC groups(P<0.05). The capacity of stimulating T cells was determined by allogeneic IL-15/lysate-DC vaccines assay, the IL-15/lysate-DC vaccines was better than the dendritic cells vaccines transfected with tumor antigens(P<0.05), In CTL assay, The capacity of killing tumor cells, the IL-15/lysate-DC vaccines was better than Lysate-DC vaccines and GFP/Lysate-DC vaccines(P<0.05).Conclusion: The recombinant advenovirus plasmid encoding mIL-15 gene was constructed successfully. It was effected to IL-15/lysate-DC vaccines in stimulating DC maturation and keeping expression of the costimulation molecules hight. It was effected to IL-15/lysate-DC succes in the capacity of stimulating T cells and killing tumor cells of CTL.