Effects Of Expression Of Tk Gene On Adult Osteosarcoma U-2Cells In Vitro

ObjectiveTo study the mechanism of bystander effect in the killing effect of GCV combined with suicide gene tk on human osteosarcoma cells.MethodsConstruct the recombinant plasmid pIRES-EGFP-tk., Then Preparat and transform the recombinant plasmids into E.coli BL21cells,Identify sequecing,extract,purificat the recombinant plasmid DNA, Culture and transfect human osteosarcoma cell line U-2OS, cell circle analysis and under fluorescence microscopy observe cell growth features, After transfection,add the selective culture medium to the TK (-) U-2OS and observe the bystander effect.ResultsSuccessfully constructed recombinant plasmids pIRES-EGFP-tk containing tk cDNA fragments. Through PCR, enzyme electrophoresis and other methods to identify the size and position of plasmids, the result was consistent with the experimental design. Observed under fluorescence microscope U-2OS (TK) showing green fluorescence. The selective culture medium of U-2OS TK/GCV had an obvious effect to suppress the cell multiplication,but when filtrated with0.2μm membrane, its effect disappeared.ConclusionsSuccessfully constructed recombinant plasmids pIRES-EGFP-tk containing tk cDNA fragments.The osteosarcoma U-2OS cells are sensitive toTK/GCV system and have significant bystander effect,which might have therapeutic potentials for osteosarcoma.