Study of cellular endocytic activity during ultrasound combined with microbubbles enhanced gene transduction of adeno-associated virus / 中华超声影像学杂志

ABSTRACT

Objective To explore the adeno-associated virus (AAV)gene transduction and cellular endocytosis mediated by ultrasound combined with microbubbles in two types of cells.Methods HeLa and NIH/3T3 cells were infected by rAAV2-EGFP at a concentration gradient to get the optimal concentrations for enhancement.At these concentrations,HeLa and NIH/3T3 cells were infected by rAAV2-EGFP mediated by ultrasound combined with microbubbles.The gene transduction efficiency were observed and measured by fluorescence microscopy and flow cytometry at 48 h after treatment.The cell viability was tested by CCK-8.The number and distribution of cellular clathrin-coated endocytic pits were observed by confocal fluorescence microscopy and transmission electron microscopy on 45 min after treatment.Results The optimal concentrations for HeLa and NIH/3T3 cells were 2000 v.g./cell and 10000 v.g./cell.Ultrasound combined with microbubbles significantly enhanced the transduction efficiency of rAAV2-EGFP (P 0.05 ).Confocal fluorescence microscopy and transmission electron microscopy demonstrated that clathrin-coated endocytic pits were more obviously increased in ultrasound combined with microbubbles mediated AAV transduction group than AAV transduction group. Conclusions Ultrasound combined with microbubbles can efficiently enhance the gene transduction of AAV,whose cellular transportation depends on cellular endocytosis,in two types of cells.Stimulating cellular endocytosis might be one of the mechanisms of enhanced cellular transportation of AAV mediated by ultrasound combined with microbubbles.