Objective To construct ultrasound targeted microbubble destruction (UTMD) combined with a polyethylenimine/pEGFPuclear localization sequence (PEI/DNA/NLS) complex gene delivery system and to evaluate the transfection efficiency of enhanced green fluorescent protein (EGFP) gene delivery to 293T cells by this system.Methods The formation of PEI/DNA/NLS complexes and the protective effects of PEI/NLS were verified by gel electrophoresis.Solutions of plasmid DNA,PEI/DNA complexes,PEI/DNA/NLS complexes,SonoVue/DNA,SonoVue/PEI/DNA complexes,and SonoVue/PEI/ DNA/NLS complexes were transduced into 293T cells via ultrasound irradiation.The expression of green fluorescent protein was observed using an inverted microscope and the transfection efficiency was detected by flow cytometry after a 24 h in vitro incubation.Cell activity was detected using a CCK-8 assay.Results Gel electrophoresis confirmed the formation of PEI/DNA/NLS complexes and showed that PEI/NLS had protective effects on DNA but only for a limited time.Inverted microscope observations showed more green fluorescent protein could be expressed under the joint action of the PEI/DNA/NLS complexes with UTMD as well as demonstrated a higher transfection efficiency by flow cytometry analysis.Further,the activity of the cells detected by CCK-8 was greater than 80％.Conclusions This study successfully constructs UTMD combined with a PEI/DNA/NLS complex gene delivery system,demonstrating that the transfection efficiency of the system is higher than that of UTMD + PEI or PEI/DNA/NLS complexes alone,without subsequently increasing the rate of cell injury.It can potentially be used for the clinical application of gene transfection.%目的 通过超声靶向破碎微泡技术与阳离子聚合物/目的基因质粒DNA/核定位信号(PEI/DNA/NLS)复合物联合构建新型基因转导体系,并评价用该体系转染293T细胞的转染效率.方法 用琼脂糖凝胶试验检测PEI/DNA/NLS复合物是否构建成功以及PEI/NLS对质粒DNA是否存在保护作用.利用超声基因转染仪作用于质粒DNA、PEI/DNA复合物,PEI/DNA/NLS复合物,声诺维微泡(SonoVue) /DNA,SonoVue/PEI/DNA复合物以及SonoVue/PEI/DNA/NLS复合物的六组293T细胞30s后,于细胞培养箱内培养24 h后,分别通过倒置荧光显微镜和流式细胞仪来观察绿色荧光蛋白的表达情况及转染效率.同时使用CCK-8试剂检测超声靶向破碎微泡(ultrasound targeted microbubble destruction,UTMD)联合PEI/DNA/NLS复合物的基因转导体系转染293T细胞后的细胞活性.结果 ①利用静电吸附作用成功构建了PEI/DNA/NLS复合物,琼脂糖凝胶电泳实验证明PEI以及NLS具有保护DNA不被核酸内切酶降解的作用,但此保护作用有时间限制.②UTMD+ PEI/DNA/ NLS复合物组在体外介导EGFP基因的转染效率优于UTMD+ PEI/DNA复合物组以及PEI/DNA/NLS 复合物(P＜0.05).③超声波的机械损伤以及PEI的化学毒性会造成细胞的损伤,但是各组之间细胞的活性差异无统计学意义.结论 UTMD联合PEI/DNA/NLS复合物构建新型基因转导体系可以提高基因的转染效率,从而提高基因治疗的效率.该基因转导体系有望为成临床基因治疗提供新的高效转染技术.
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