| Objective: To study granulocyte-macrophage colony stimulating factor (GM-CSF)gene-modified dendritic cell (DC), which mobilized into the peripheral blood by injection ofmacrophage inflammation protein-1α(MIP-1α) and the anti-tumor effects induced by DCvaccine expressing GM-CSF in mice.Methods: (1) 615 mice were injected with MIP-1αvia the tail vein. After MIP-1αinjection, the peripheral blood mononuclear cells (PBMCs) were prepared from theperipheral blood. F4/80~-B220~-CD11c~+ DC Precursor were sorted from these PBMCs byFACS. Then F4/80~-B220~-CD11c~+ cells were cultured into DC by cytokines. DC weretransferred AdGM-CSF gene at different radios of multiplicity of infection (MOI) todetermine the optimal gene transfection conditions, and detecting the expression ofGM-CSF after transfection. (2) The variation of GM-CSF gene-modified DC wereanalysed by morphological observation, phenotype analysis, and mixed lymphocytereaction (MLR). (3) GM-CSF gene-modified DC were loaded with mouse forestomachcarcinima (MFC) antigen obtained by frozen and thawed to construct DC vaccineexpressing GM-CSF (GM-CSF-DC-MFC Ag). (4) To establish the solid tumor model,groups of 615 mice were injected with MFC cells subcutaneously in the abodminal wall.GM-CSF-DC-MFC Ag were immuned before and after the challenge of MFC cells, thenwe observed the tumor size and the survival of mice which could detect the protective andtherapeutic effect of DC vaccines.Results: (1) F4/80~-B220~-CD11c~+ cells increased in the circulation 8 hour afterMIP-1αinjection, then gradually reached the peak level 48 hour after injection. (2) ELISAresults showed that after GM-CSF gene modification, DC could produce high level ofGM-CSF. When AdGM-CSF was transferred into DC (MOI=100), GM-CSF level inculture supernatants reachs saturation. (3) After GM-CSF gene-modification, DC tend tomore maturated through morphological observation and phenotype analysis. At the same time, the capacity of activating allogeneic T lymphocytes proliferation was enhancedgreatly. (4) The 615 mice were injected with MFC cells subcutaneously after immunizingDC vaccine. 22 days after immunization, the tumor size of the experimental group was(2.17±.090) cm~3; the tumor size of DC-MFC Ag and LacZ-DC-MFC Ag control groupswere (7.95±1.43) cm~3 and (8.26±1.64) cm~3 (compared with the experimental group, P＜0.05); while tumor growth of DC control group was much faster (compared with theexperimental group, P＜0.01); Kaplan-Meier survival curves show that the survival of theexperimental group mice were much longer. (5) Five days after the MFC cells challenge,615 mice were subsequently injected with GM-CSF-DC-MFC Ag. The tumor size of theexperimental group was (2.56±0.72) cm~3. the tumor size of DC-MFC Ag andLacZ-DC-MFC Ag control groups were (9.03±1.58) cm~3 and (9.47±1.45) cm~3 (comparedwith the experimental group, P＜0.05 ); while tumor growth of DC control group wasmuch faster (compared with the experimental group, P＜0.01)Kaplan-Meier survivalcurves show that the survival of the experimental group mice were much longer, five miceof which were still alive 70 days after the MFC cells challenge. The difference between theexperimental group and the control groups is statistically significant (P＜0.01).Conclusion: (1) After GM-CSF gene modification, DC could produce high level ofGM-CSF. (2) After GM-CSF gene-modification, DC tend to more maturated, and thecapacity of activating allogeneic T lymphocytes proliferation was enhanced greatly. (3)The DC vaccine expressing GM-CSF could stimulate T cells for obviously protective andtherapeutic anti-gastric carcinoma effects on MFC cells loading mice in vivo. (4) Theefficiency of anti-gastric carcinoma immunity induced by GM-CSF-DC-MFC Ag weremuch more potent than DC-MFC Ag.
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