| Objective: PC3-DC fusion cells were made by human prostate cancer cells(PC3)and dendritic cells(DC)fusion cells with the use of PEG,the fusion cells were modificated by B7.1/GM-CSF protein and observed the specific antitumor immune effect in vitro.Methods: 1.Construction of GM-CSF and B7.1/GPI plasmid and preparation of B7.1/GPI protein.B7.1,GPI and B7.1 extracellular domain primers were designed by Primer Premier software.PCDNA3.1-GM-CSF plasmid and PCDNA3.1-B7.1/GPI plasmid were builded and transfected to DH5 a cells.PCDNA3.1-B7.1/GPI plasmid and PSV2-DHFR plasmid were transfected into CHO/DHFR-cells,cultured in DMEM:F12=1:1(10% fetal bovine serum,1×10-6 mol/LG418),B7.1/GPI-CHO cells which stable express B7.1/GPI protein were screened.A lot of B7.1/GPI protein were build by MTX pressure screening,the expression of B7.1/GPI in CHO/DHFR cells were tested by Western and immunofluorescence assay.2.Preparation and morphological observation of B7.1/GM-CSF modified PC3-DC fusion vaccine.PCDNA3.1-GM-CSF plasmid was transfected into PC3 cells by the transfection kit,and the PC3 cells stably expressing GM-CSF were screened by G418,Western blot and immunofluorescence.peripheral blood mononuclear cells were separated by lymphocyte isolation liquid.The DC and GM-CSF-PC3 cells were fusioned at the rate of 1:5,the two kinds of cells were cultured in 1640 medium(10%fetal bovine serum,500ng/ml GM-CSF)for 14 d,and be screened by magnetic beads.The fusion cells were resuspended in RPMI 1640 medium(5 × 106 /ml),B7.1/GPI protein was added in the culture medium(10ug/ml),cells were intermittent concussion for 4 hours,then cells were cultured in 24 well plates.The biological characteristics of fusion vaccine and antitumor effect of tumor vaccine against prostate cancer in vitro.The growth of the fusion vaccine was observed and the cell growth curve was depicted,then the increment ability of fusion cells,DC cells and PC3 were compared.3.The ability that stimulated T cells secreting IFN-gamma and T cell proliferation of4 kinds fusion cells and DC were compared.The ability of stimulate CTL to kill PC3 cells of 4 kinds of fusion cells and DC were detected by LDH.Results: 1,CHO cells which can secrete B7.1/GPI protein were selected by MTX and GM-CSF-PC3 cells were successful prepared;DC and PC3 cells are effectively fused by PEG.Screening of fusion cells can be effectively screened by magnetic beads.High expression of DC and E-cadherin markers of fusion cells.2,B7.1/GM-CSF-PC3-DC had the strongest ability to stimulate T cells proliferation and secretion of the 4 kinds of fusion cells,followed by GM-CSF-PC3-DC,B7.1-PC3-DC two kinds of fusion vaccine,the worst for PC3-DC fusion vaccine.4 kinds of fusion cells were significantly higher than DC,the difference was significant(P<0.05),B7.1/GM-CSF-PC3-DC had the strongest ability stimulate CTL to kill PC3 cells of the 4 kinds of fusion cells,followed by GM-CSF-PC3-DC,B7.1-PC3-DC two kinds of fusion vaccine,the worst for PC3-DC fusion vaccine.4 kinds of fusion cells were significantly higher than DC,the difference was significant(P<0.05)Conclusion: 1,B7.1/GPI-CHO/DHFR cells expressed B7.1/GPI protein in a large numbers,and B7.1/GPI protein stably anchored to the fusion cell surface;The GM-CSF-PC3 cells can express GM-CSF,PEG fusion technology can successfully induce DC and GM-CSF-PC3 fusion and a more pure fusion vaccine can be obtained by magnetic bead screening;fusion vaccine expression DC and PC3 surface antigen.2,PC3-DC fusion vaccine to stimulate T cell proliferation and IFN-secretion was significantly stronger than that of DC,and the PC3-DC fusion vaccine induced effector cell specific killing ability of CTL PC3 was significantly stronger than that of DC,and fusion vaccine was modified by B7.1/GM-CSF on T cell proliferation and IFN-secretion ability and induced CTL effect cell specific killing ability of PC3 was stronger than that fusion vaccine without modification of B7.1/GM-CSF.3,PC3-DC fusion vaccine has obvious killing effect of tumor cells,and B7.1/GM-CSF as a specific modification,can significantly improve the killing effect on tumor fusion vaccine,immunotherapy has become an effective means for prostate cancer. |
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