In vitro study of induction of specific cytotoxic T lymphocytes by the dendritic cells co-transfected with pancreatic cancer-associated antigen MUC1 and survivin mRNA / 中华胰腺病杂志

ABSTRACT

Objective To investigate the ability of induction of specific cytotoxic T lymphocytes (CTL) stimulated by dendritic cells (DCs) co-transfected with MUC1 and survivin mRNA of human pancreatic cancer,and to provide the experimental basis for the treatment of human pancreatic cancer with multi-epitope DC vaccine.Methods DCs were isolated and cultured from peripheral blood mononuclear cells (PBMCs) of 6 patients with pancreatic cancer.Human pancreatic cancer cell line MiaPaCa-2 was routinely cultured,after being transcripted and amplified by RT-PCR,MUC1 and survivin mRNA were co-transfected or individually transfected into DCs by electroporation,and they were named as DC-MUC1,DC-survivin,DC-MUC1 + survivin.The expression of MUC1 and survivin mRNA in DCs were detected by real-time PCR.The survival rate of transfected DCs were determined by MTT method.The lymphocyte proliferation ability was evaluated by mixed cell culture method.The Th1 cytokine releasing of antigen-specific CTLs were measured by ELISA assay.Results Mature DCs were obtained,the positive expression rates of surface markers CD40,HLA-DR,CD83 and CD86 were 34.31％,50.21％,89.17％ and 73.62％,respectively.The expression amount of MUC1 mRNA of DC-MUC1 was 36.24 ± 5.17,and the expression amount of survivin mRNA of DC-survivin was 34.53 ± 4.02,while the expression amounts of MUC1,survivin mRNA of DC-MUC1 + surviving were 31.79 ±4.26 and 14.67 ± 2.96,which were significantly lower than that in individual transfection group (P ＜ 0.05).The survival rate of DC-MUC1 + surviving was decreased in a time dependent manner,which was significantly lower than that in individual transfection group (about 50.21％ vs 80％ at 24 h,P ＜0.05).When DC/T cells ratio was 1∶ 10,1∶ 20,the autologous T cell proliferation index of MUC1 and survivin mRNA in co-transfection DC group was significantly higher than that in individual transfection group (P ＜ 0.05) ;when DC/T cells ratio was 1∶ 40,1∶ 80,the difference of proliferation index was not statistically significant.When DC/T cells ratio was 1∶ 10,after 14 d culture,the expressions of IL-2 in DC-MUC1,DC-survivin,DC-MUC1 + surviving were (892.73 ± 32.9),(713.62 ± 56.37),(1884.37 ± 95.21) pg/ml,and the expressions of granzyme B were (501.62 ± 12.30),(203.84 ± 12.55),(1193.15 ± 86.04) pg/ml ; and the expressions of IFN-γ were (981.50 ± 47.82),(696.05 ± 41.66),(2237.94 ± 189.55) pg/mL.The corresponding values in DC-MUC1 + surviving group were significantly higher than those in individual transfection group (P ＜ 0.05) ; while the difference of IL-10 was not statistically significant.Conclusions DCs co-transfected with MUC1 and survivin mRNA have a stronger ability to stimulate specific CTL in vitro than individual antigen loaded DCs.