The Influence Of VEGF Targeted Gene Silencing Of T24 Cells In Bladder Cancer On Dendritic Cells’ Differentiation, Maturation And Immunity

objective: The VEGF(vascular endothelial growth factor,VEGF)gene of T24 cells in Bladder cancer will be targeted silenced by using the RNAi(RNA interfering,RNAi)technology.And the supernatant after culturing T24 cells will be add into the dendritic cells(DCs)which derived from PBMC(Peripheral blood mononuclear cells)to observe the influence of the VEGF secreted by bladder cancer on dendritic cells’ differentiation,maturation and immunity.Meanwhile,discuss the mechanism of VEGF in the bladder cancer’s microenvironment on immune escape of the bladder cancer and on anti-tumor effect of the DCs.The supernatant is to simulate the microenvironment of the bladder cancer.Methods: Three groups were set up in the experiment,which are the infected group,the empty vector group and the uninfected group.We construct a lentiviral vector named LV-VEGFA-RNAi for gene silencing targeting VEGF as the infected group and a lentiviral vector named LV-CON without any valid sequences as the empty vector group.The two different kinds of lentiviral vectors will infect the T24 cells of the corresponding groups.And the uninfected group will accept no intervention measures.The T24 cells after infection will be observed by the fluorescence microscope to confirm that infection is successful and the uninfected T24 cells will be observed by the inverted microscope.We use the Reverse Transcription Polymerase Chain Reaction(RT-PCR),the Real-time Quantitative PolymeraseChain Reaction(qPCR)and the enzyme linked immunosorbent assay(ELISA)to detect the expression of VEGF’s mRNA and protein,respectively.Then the immature DCs will be co-cultured with the supernatant of the T24 cells of all the groups mentioned before.The flow cytometry will be used to detect CD1 a,CD83 as the maturation marker and CD86 etc.as the immunity marker of the DCs.The result will be analyzed by SPSS17.0 statistics software.Results: In spite of some injury,the T24 cells of two groups were infected by the lentiviral vectors,successfully.The expression of VEGF’s mRNA and protein of T24 cells in the infected group was inhibited obviously(P<0.05)compared with the result of the empty vector group and the uninfected group while the results of the empty vector group and the uninfected group had no statistical significance(P>0.05)when compared with each other.After co-culture of the supernatant and the DCs,DCs of the infected group had an obviously increase on CD1 a,CD83 and CD86 compared with DCs of the empty vector group and the uninfected group while the results of the empty vector group and the uninfected group when compared with each other stayed no statistical significance(P>0.05)as mentioned above.Conclusion: VEGF is a very important immunosuppressive factor in tumor microenvironment.And dendritic cell is found as the most powerful and the only one antigen-presenting cell(APC)which can activate the initial T lymphocyte of the body,which plays a very important role in the anti-tumor immune response of the body.By the results of this experiment,we can know that bladder cancer cells secrete VEGF which can inhibit the differentiation,maturation and immunity of DCs,which mayhave relationship with the immune escape of the bladder cancer.VEGF targeted gene silencing by RNAi has advantages to the growth and immunity of DCs,which may strengthen the anti-tumor capacity of the DCs by repairing their damaged immune monitoring function.