Anti-tumor Effect Of Dendritic Cell Vaccines Transfected With Total RNA Of Human Lung Adenocarcinoma Cell Through Electroporation In Vitro

Objectives: To make definite the practicability of sorting and cultivating DCs (dendritic cells) from rhG-CSF(human recombination granulocyte colony stimulating factor ) mobilized peripheral blood, to explore the characteristics and the capability of specifically inducing the anti-tumor immunological effect of dendritic cells (DCs) transfected with human lung adenocarcinoma cell line A549 total RNA through electroporation.Methods: Induced DCs from the peripheral blood mononuclear cells with rhGM-CSF, rh IL-4.Then, DCs transfected with total RNA extracted from A549 cells through electroporation. Cytotoxic T Lymphocyte(CTLs) were induced by cocultured with transfected DCs. All DCs were divided into three groups: non-transfected DCs, PBS-transfected DCs and DCs transfected with A549 total RNA. Surface markers on DCs ,T cell and CTLs were detected by flow cytometry.RNA transfection efficiency was verified by RT-PCR.T cell proliferation was judged by MTT.Enzyme-linked immunoassay( ELISA) was uesed to detect the secretion of IL-12 and IFN-γ.The specific cytotoxicity was measured by lactate dehydrogenase(LDH) content.Results:1)The A549 total RNA can be transfected through electroporation successfully and DCs can express the tumor antigen.2) The surface markers CD40, CD80, CCR-7, CD83, HLA-DR on total RNA-transfected DCs were highly expressed compared with the non-transfected DCs and PBS-transfected DCs(P<0.05).3)The ratio about surface markers CD8 on T cell which stimulated by RNA-transfected DCs increased significantly.4) The expressions of IL-12 and IFN-γwere dramatically increased in RNA-transfected DCs than non-transfected DCs and PBS-transfected DCs(P<0.05). 5) The RNA-transfected DCs can stimulate T cell to proliferate effectively.And it induced T-cell change into tumor-specific CTLs which can kill the A549 cells specifically. However non-transfected DCs and PBS-transfected DCs had no significant effect(P<0.05).Conclusion: We can sort and induce DCs with typical morphology and immune phenotype from rhG-CSF mobilized peripheral blood by density gradient centrifugation method,DCs transfected with human lung adenocarcinoma cell A549 total RNA through electroporation can express the tumor-associated antigen. And it can induce tumor-specific immune response effectively in vitro.