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Objective To investigate the transfection efficiency of combining ultrasound targeted microbubble destruction (UTMD) and classic nuclear localization signal (cNLS) peptide for facilitating targeted gene into nucleus.Methods The pEGFP was transfected into the 293T cells by different ways.This study was divided into 4 groups, control group : UTMD +pEGFP;cNLSgroup: UTMD +pEGFP +cNLS;mutativegroup : UTMD +pEGFP +mNLS; and retardantWGA group:UTMD +pEGFP+cNLS +WGA.The NLS was labeled by FITC and pEGFP was maked by Cy3.6h after transfection,the rate of plasmid DNA into cells was detected as the percentage of Cy3 positive cells by flow cytometry,the distribution of Cy3 fluorescent plasmid in cell organelles was observed by laser confocal microscope, the rate of fluorescence intensity into nuclear was calculated by the percentage of Cy3 fluorescence intensity in the nucleus to the whole cell.48h after transfection, the transfection efficiency was detected by flow cytometry, the survival rate of cells was measured by CCK8.RT-PCR and Western Blot were used to detect the mRNA and protein expression of the relative quantity, and compared the differences of the above indicators in 4 groups to evaluate the enhancement effects of cNLS during UTMD mediated gene transfection.Results (1)At 6h after transfection,almost all green fluorescence showed in the nucleus in cNLS group,it appeared in both the cytoplasm and nucleus in mNLS group,but mostly appeared in the cytoplasm and almost none nucleus in WGA group.(2) 6h after transfection, the rate of pEGFP into the cells were (61 ± 11)%, (80± 10)%, (55±9)% and (58±10)%;the rate of pEGFP into the nucleus were (20±4)%, (50±11)%, (18±3)% and (10±3)% in 4 groups respectively.The rates were highest in cNLS group and lowest in WGA group.There was no significant difference between mNLS group and control group.(3) 48h after transfection,the cell activity were greater than 80% in all groups;the transfection efficiency, the relative quantityof mRNA and protein were highest in cNLS group and lowest in WGA group, the difference was statistically significant(P<0.05).Conclusion The UTMD combining cNLS can promote pEGFP into the nucleusfor improving the transfection efficiency.