Construction of Antisense VEGF_(121) cDNA Eukaryotic Expression Plasmid and Its Effect on Human Bladder Cancer Cell

Purpose To construct and identify eukaryotic expression recombinant plasmid bearing human VEGF121 cDNA and evaluate the effect of antisense gene therapy on bladder cancer. Methods We inserted VEGF121 into eukaryotic expression vector pcDNA3 and constructed pcDNA3-As- VEGF121. Restriction endonuclease analysis was used to confirm the reverse orientation of the VEGF cDNA. The recombinant plasmid was transfected into human bladder cancer cell line T24 and the positive done was screened by G418. VEGF mRNA and protein expression of T24 cells before and after transfection were detected by reverse transcription PCR and ELISA, respectively. The biological characteristics of T24 cells before and after transfection were also inspected. Results The pcDNA3-As-VEGF121 recombinant plasmid was obtained. VEGF expression was blocked by antisense VEGF RNA. The done formation ability of single cell was decreased after transfection. The obvious apoptosis and inhibition of proliferation of T24 cells after transfection were observed by electron microscope,flow cytometer and MTT methods. Conclusions The successful construction of antisense VEGF121 eukaryotic expression recombinant plasmid and the biological characteristics of the transfected cell may provide the basis for the development of antiangiogenic gene therapy to bladder cancer.