Anti-tumor Effect Of Dendritic Cell Vaccines Transfected With Total RNA Of A549 Cells In Vitro Study

Objectives: 1) To make definite the practicability of sorting and cultivating DCs (dendritic cells) from rhG-CSF(human recombination granulocyte colony stimulating factor ) mobilized peripheral blood. 2) To study the feasibility of preparing DCs vaccine that transfected by liposome with tumor cell A549 total RNA and the optimization of experimental conditions. 3) To observe the DCs vaccines'anti-tumor immune response capability in vitro.Methods: Collected fifteen doners'blood of peripheral blood stem cell transplantation, the doners were given rhG-CSF 10μg·kg-1·d-1 five days as routine, on the five day,collected their blood through blood seperation instrument. Using density gradient centrifugation method, the peripheral blood mononuclear cells (PBMCs ) were isolated from fresh peripheral enriched blood, then obtain monocyte via making PBMCs adherent, DCs were generated from monocyte and transfected with total RNA extracted from A549 cells for preparing vaccine, and cocultured with T cells to induce specific cytotoxic T lymphocytes (CTLs). DCs were divided into three groups to carry out the experiments. They were DCs transfected by liposome with tumor cell A549 total RNA, non-transfected DCs and liposome-transfected DCs. Surface markers on DCs were detected by flow cytometry; the secretion of IL-12 and IFN-γwas detected by enzyme-linked immunosorbent assay (ELISA); T cell proliferation was judged by 3H-TdR incorporation assay; the specific cytotoxicity was measured by lactate dehydrogenase (LDH) content. Results:1)Surface markers on total RNA-transfected DCs were expressed with remarkably higher levels than non-transfected and liposome-transfected DCs(P<0.05), CD40 expression rate in these three groups were (69.01±7.67)%, (19.28±3.51)% and (39.62±2.72)%; CD80 expression rate in these three groups were (74.39±6.46)%,(15.48±3.03)% and (25.70±2.92)%; CD83 expression rate in these three groups were (81.79±4.61)%, (13.29±2.34)% and (31.42±1.97)%; HLA-DR expression rate in these three groups were (76.53±5.13)%, (49.23±4.05)% and (29.94±3.53)%; 2)the secretion of IL-12 and IFN-γwas dramatically increased in RNA-transfected DCs than non-transfected and liposome-transfected DCs(P<0.05), IL-12 was(543.24±68.33)pg/ml, (77.11±27.65)pg/ml and (167.78±31.84)pg/ml;IFN-γwas (122.95±32.84)pg/ml, (41.06±10.97)pg/ml and (56.08±15.96) pg/ml; 3)the counts per minute (CPM) was more higher in total RNA-transfected DCs (7 437±720), non-transfected DCs(2 207±296) and liposome- transfected DCs(2 443±314), and positive control (16 739±91); Specific CTLs induced by RNA-transfected DCs exhibited a higher level of cytotoxicity(58.55±8.27)% than non-transfected DCs(36.03±4.00)% and liposome- transfected DCs(33.47±8.21)%, P<0.05.Conclusion: 1) We can sort and induce DCs with typical morphology and immune phenotype from rhG-CSF mobilized peripheral blood by density gradient centrifugation method. 2) DCs transfected by liposome with tumor cell A549 total RNA can be used to prepare vaccine. 3) DCs vaccines can effectively stimulate T cell proliferation and kill tumor cells.