The Inhibitory Effect Of Suicide Gene Transfection Combined Multi-Tissue-Specific Cytokine Eukaryotic Expression Vector For Bladder Cancer

Objective Explore five kinds of multi-joint suicide gene expression of cytokines urinary tissue-specific gene eukaryotic expression vector pBud-UPII-TK/CMV-IL-2pBud-UPII-TK/CMV-TNF-a, pBud-UPII-TK/CMV-TRAIL, pBud-UPII-TK/CMV-IT pBud-UPII-TK/CMV-ITR’s inhibition for human bladder cancer5637.Methods Transfect the Dual promoter vector pBud-UPII-TK/CMV-IL-2, pBud-UPII-TK/CMV-TNF-a, pBud-UPII-TK/CMV-TRAIL, pBud-UPII-TK/CMV-IT, pBud-UPII-TK/CMV-ITR into bladder cancer cells5637, which we built and parallel sequencing, PCR and restriction endonuclease digestion proved before, screened Zeocin resistant clones by resistance marker, detection the expression of five vector IL-2, TNF-a, TRAIL protein expression by westen blot, compared it with untransfected bladder cancer cells5637transfected with empty vector and target cells pBud4.1; analysis IL-2/TNF-a and TRAIL mRNA expression before and after transfection and transfection of different carriers, Let untransfected bladder cancer cell5637as a control, compare the difference expression of its mRNA; MTT was used to detect the difference between five target cells, non-transfected and transfected with the empty vector pBud4.15637cells’s inhibition rate, And observe the effects of inhibition rate five vector on bladder cancer5637cell after adding25ug/ml GCV; Using flow cytometry detect apoptosis rate’s difference between five target cells and untransfected bladder cancer cell5637, and its results were Statistical analysised.Results Westen blot results showed that after five co-transfected expression vectors expressing target cells corresponding cytokine protein was significantly higher than non-transfected cells and the empty vector5637; RT-PCR confirmed in5637after transfection cells were IL-2, TRAIL TNF-a mRNA expression was significantly increased; flow cytometry results after five tips were transfected with an expression vector, the apoptotic rate was significantly higher than the group transfected with empty vector and non-transfected group (P<0.01); MTT the results also show that co-expression vector transfected cell proliferation was significantly inhibited (P<0.01), the inhibition rate was significantly increased after adding25ug/ml GCV (P<0.01).Conclusion UPII promoter-driven HSV-TK gene, with IL-2, TNF-a, TRAIL, IL-2-TNF-a (IT), IL-2-TRAIL (ITR) these five dual promoters reorganization eukaryotic expression plasmid, which can effectively accelerate apoptosis in bladder cancer cell5637, after adding GCV,the bladder cancer cell5637inhibition rate significantly increased. This study laid the foundation of Suicide gene and multi-Cytokine gene combined with urinary tissue-specific cytokine eukaryotic expression vector used in the treatment of bladder cancer.