| Objective:To construct the eukaryotic expression vector PcDNA3.1-IE-ARG1 and study the arsenic resistance of ARG1 Gene after transfecting 293T cell.Methods:293T cells were transfected with the recombinant plasmid by liposome,then examined by laser scanning confocal microscopy (LSCM)and the expression of recombinant ARG1 gene mRNA was examined by real-time PCR; 48 hours acute arsenite toxicity test was performed to assay cells live rate by MTT;Three different groups of cells were exposed to sodium arsenite (1,4,8μmol/L) for 24 hours,arsenite accumulation, to determined the arsenite accumulation and efflux by atomic absorption spectrophotometry. The cells were exposed to sodium arsenite (0,4,8μmol/L) for 48 hours, to determined the intracellular concentration of GSH and GST activity by DTNB, the expression of MRP2 was determined by Western-blot analysis.Results:The real-time PCR results showed that the recombinant plasmid were successfully transfected into 293T cells.48 hours acute arsenite toxicity test shows that the live rate of the cells was inhibited by sodium arsenite in time-and dose-dependant manner, but compared with empty plasmid group and untransfected group,the live rate of the ARGlgene group was much higher when the cells were exposed to lower sodium arsenite (≤8μmol/L). When cells were exposed to sodium arsenite with 1,4,8μmol/L for 24 hours, arsenite contents of the cells of the ARG1gene group was lower,and the efflux of arsenic was higher than that of empty plasmid group and untransfected group cells (P<0.05). When cells were exposed to sodium arsenite in 0,4,8μmol/L for 48 hours,intracellular concentration of GSH, GST activity and expression of MRP2 in the cells with ARG1gene group were higher than that of empty plasmid group and untransfected group cells (P<0.05), and increased along with the increased arsenic.Conclusion:ARG1 gene increased arsenic resistance of cell when the expression of ARG1 gene was higher in 293T cell. |
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