In vitro study on the induction of antigen-specific cytotoxic T lymphocytes by fusion of dendritic cells with pancreatic canec r MiaPaCa-2 cells / 中华胰腺病杂志

ABSTRACT

Objective To investigate the ability of inducing antigen-specific cytotoxic T lymphocytes (CTL) stimulated by dendritic cell (DC) fused with MiaPaCa-2 cells in vitro.Methods DC were isolated and cultured from peripheral blood mononuclear cells (PBMCs).50%PEG and 10%DMSO were used to fuse MiaPaCa-2 cells and DC, and DC co-cultured with MiaPaCa-2 cells and DC alone served as control .The fusion efficiency was assessed by flow cytometry ( FCM) and DC-MiaPaCa-2 hybrids were identified as PE-MUC1/FITC-CD86 double positive cells . The survival rate of DC was determined by MTT method . The lymphocyte proliferation stimulated by DC in vitro was evaluated by mixed cell culture with DC in different ratios of 1∶10, 1∶20 and 1∶80.IL-2, IL-10, granzyme B and IFN-γreleased by antigen-specific CTLs were measured by ELISA assay.Results The fusion rate in DC fused with MiaPaCa-2 cells ( fused cells ) was ( 42 .30 ± 7.30)%, which was higher than (7.21 ±1.06)% in DC co-cultured with MiaPaCa-2 cells ( co-cultured cells).The cell viability of DC, co-cultured cells and fused cells was >95.0%, 85.0% and 62.8%, and fused cells had greatly lower cell viability than DC and co-cultured cells (P0.05).At the co-culture ratio of 1∶10, IL-2 secreted by CTL in DC, co-cultured and fused cells was (27.30 ±5.21 ),(897.44 ±93.05),(2 243.80 ±381.46) ng/L; IL-10 was (19.55 ± 2.05), ( 424.60 ±108.25 ), ( 706.53 ±161.29 ) ng/L; Granzyme B was ( 16.23 ±1.23 ), ( 451.07 ± 120.50),(1327.77 ±205.15)ng/L;IFN-γwas (30.11 ±4.32)、(982.00 ±124.68)、(2421.04 ±488.50) ng/L.Cytokines from the antigen-specific CTL induced by DC fused with MiaPaCa-2 cells were significantly higher than those by DC and DC co-cultured with MiaPaCa-2 cells ( all P<0.05).Conclusions The fusion of DC and pancreatic cancer MiaPaCa-2 cells could stimulate tumor antigen-specific CTL in vitro .