Experimental Study On The Effect Of Dendritic Cell Vaccine Targeting To Interference Vascular Endothelial Growth Factor On Human Bladder Cancer Cell Line T24

Objective: We will targeted down-regulation of T24 bladder cancer cells VEGF(vascular endothelial growth factor,VEGF)expression by using RNA interference(RNA interference,RNAi)technology,and observe the differentiation and maturation influence of DC(dendritic cell,DC)precursor cells by reducing the expression of VEGF in bladder cancer T24 cells.The DC vaccine will be prepared by using the whole antigen of bladder cancer T24 cells.we will investigate the killing effect of the special cytotoxic T lymphocytes on bladder cancer cell line T24 mediated by the DC vaccine after down-regulation of VEGF.Methods: We use lentiviral vector to construct LV-VEGFA-RNAi,and transfect into bladder cancer cell line T24 to down regulate the expression of VEGF.The transfection experiment was divided into 3 groups: transfection group(LV-VEGF),negative transfection group(LV-CO)and untransfection group(CO).The growth and expression of fluorescence of the cells were observed by inverted microscope and fluorescence microscope.The level of mRNA VEGF and VEGF protein of T24 bladder cancer cells in each group wil be detected by using qRT-PCR and ELISA.We prepare three groups DC vaccine: control group(routine medium),interference group(25% volume fraction of transfection group culture supernatant)and no interference group(25% volume fraction of untransfection group culture supernatant).Using rh IL-4(recombinant human interleukin-4,rhIL-4)and rhGM-CSF(recombinant human granulocyte-macrophage colony stimulating factor,rhGM-CSF)to induce the differentiation of DC in vitro.After eighth days,the whole antigen of bladder cancer T24 cells were added to each group.Two hours later,the LPS(Lipopolysaccharides,LPS)was added to each group to stimulate the maturation of DC.On the fourteenth day,the morphology of DC was observed by inverted microscope.The DC phenotype of CD1 a,CD83 and CD86 were identified by flow cytometry.We will separation T lymphocytes from human peripheral blood by nylon wool column and detect the CD3 of T lymphocytes by flow cytometry.The proliferation and cytotoxicity of T lymphocytes stimulated by DC vaccine were detected by MTT.The supernatant of each group was collected,and the levels of IL-10,IL-12 and IFN-γwere detected by ELISA.The SPSS 17.0 software was used to analyze the experimental data.Results: The expression of VEGF of bladder cancer T24 cells reduced successfully by transfecting the LV-VEGFA-RNAi.Compared with the untransfected group,the expression of mRNA VEGF and VEGF protein in transfected group decreased(P<0.05).The expression of mRNA VEGF and VEGF protein were no difference between the two groups in the negative transfection group and the untransfection group(P > 0.05).The phenotypes of DC vaccine were detected by flow cytometry.Compared with the control group,the DC phenotype of CD1 a,CD83 and CD86 of the interference group and the no interference group decreased differently(P<0.05);Compared with the no interference group,the DC phenotype of CD1 a,CD83 and CD86 of the interference group increased(P<0.05).The level of IL-10,IL-12 secreted by the DCs in each group were different(P < 0.05).Compared with the control group,the level of IL-12 in the interference group and interference group decreased in varying degrees,while the level of IL-10 increased in varying degrees.The proliferation of T lymphocytes stimulated by DC vaccine in vitro was different in each group(P < 0.001),while the control group had the strongest proliferation(SI:10.90±0.49)and the no interference group had the weakest proliferation(SI:8.03±0.39).Compared with the no interference group,the proliferation of T lymphocytes of interference group(SI:8.81±0.41)was stronger(P < 0.001).The cytotoxicity of T lymphocytes activated by DC vaccine on bladder cancer T24 cells was different in the interference group(46.23±1.07)%,no interference group(32.50±0.38)% and control group(54.64±0.90)%(P < 0.001).The control group had the strongest cytotoxicity and the no interference group had the weakest cytotoxicity.Compared with the no interference group,the cytotoxicity of T lymphocytes of interference group was stronger(P < 0.001).The level of IFN-γ secreted by CTL were different in the interference group(37.45±0.38)pg/ml,no interference group(21.87±0.30)pg/ml and control group(51.08±0.41)pg/ml(P < 0.001).Conclusion: The VEGF secreted by bladder cancer T24 cells inhibited maturation of DC which further inhibited the cellular immunity in vitro.Inhibiting tumor VEGF expression in vitro,which is helpful to enhance the cytotoxicity of CTL mediated by DC vaccine on T24 bladder cancer cells.