| Objective:To investigate the affects of Helicobacter pylori and gastric cancer antigen on the maturation and antineoplastic function of monocyte derived dendritic cell from human peripheral blood in vitro, and to find out a more optimal stimulation time for dendritic cell.Methods:Peripheral blood mononuclear cells were collected from human volunteers by density gradient centrifugation on Ficoll-Paque. Immature dendritic cells were generated from monocytes isolated by adherent method and cultured in RPMI-1640complete medium in the presence of rhGM-CSF (1000U/ml) and rhIL-4(500U/ml) for five days. Every three days, fresh medium, rhGM-CSF and rhIL-4were added to the cells. On day5of culture, the panel were divided into three groups. Group with the treatment of sonicated H.pylori at10μg/ml and rhTNF-a at50ng/ml after it was added gastric carcinoma cell BGC823lysate at0.1mg/ml for4hours was marked as group Hp+Ly-mDC.The group without treatment of lysate, sonicated H. pyloriand and rhTNF-a as control group(group iDC)and the group with the treatment of gastric carcinoma cell BGC823lysate and rhTNF-a was as group Ly-mDC. Mature DCs were obtained by the stimulation of iDC for8,24,48, and72h with sonicated H. pylori and rhTNF-a. Morphology of DC was observed daily by an phase contrast inverted microscope during the culture period. The phenotype CD83, CD86and HLA-DR expression on DC was determined by flow cytometry. The capacity of DC in stimulating proliferation of autologous T lymphocytes in mixed lymphocyte reaction was determined by MTT assay. The cytolytic cells’activity, induced by different DCs pulsed CTLs as effector cells, and with gastric carcinoma cells BGC823as target cells, were measured using LDH-releasing assay. The secretion of IL-12and IFN-γ in the supernatant was determined by ELISA. Results:(1) mDC was exhibited an irregular with extensive cellular protrusions and veils extending from all aspects of the cellular body. There was no difference in the morphological change of the maturation process in group Ly-mDC and group Hp+Ly-mDC.(2) The mean expression of CD83, CD86and HLA-DR molecules on denritic cells of group Hp+Ly-mDC were higher than those of group Ly-mDC,and the differences were statistically significant except for HLA-DR.The highest expression was observed in group Hp+Ly-mDC when the stimulation time for24h and group Hp+Ly-mDC for48h.(3) The capacity of group Hp+Ly-mDC in stimulating proliferation of autologous T lymphocytes reached a maximum when the stimulation time for24h (SI=3.52±0.31).(4) The cytotoxicity of specific CTL against gastric carcinoma cell in group Hp+Ly-mDC was statistically higher than those of group Ly-mDC and group iDC (p<0.01). The specific cytotoxicity were (43.41±1.85)%and (37.67±1.93)%at the24h,48h time point, this difference was significant (p<0.05).(5) The largest release of IL-12, IFN-γ in group Hp+Ly-mDC for48h of stimulation were (627.24±31.05) pg/ml and (1313.56±48.41) pg/ml, and it was significantly higher level compared to group Ly-mDC (p<0.01).Conclusion:These data clearly demonstrated that sonicated H.pylori could induce a stronger maturation and activation. The antigen presentation and antineoplastic function of dendritic cells in group Hp+Ly-mDC were superior to group Ly-mDC. The kinetics of maturation and activation of lysate, sonicated H.pylori plused DC with stimulation time could be fouded and the24h of stimulation was a more optimal. |
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