超声造影剂Levovist介导质粒GFP转染小鼠骨骼肌细胞的实验研究

目的探讨空气型微泡造影剂Levovist介导基因转染小鼠骨骼肌细胞的作用。方法采用质粒GFP作为目的基因，超声(1MHz脉冲波，20％工作周期，空间时间峰值强度1W／cm^2)结合Levovist作用于小鼠H2K成肌细胞，照射时间分别为10s、20s、30s、40s、50S、60s，流式细胞仪测定GFP阳性细胞率，台盼蓝染色测定细胞生存率。超声结合Levovist作用于小鼠胫前肌，1周后处死小鼠，荧光显微镜测定GFP阳性肌纤维数，HE染色估计肌肉破坏面积。结果超声单独作用于H2K细胞显示出较佳的增强GFP基因表达的作用，加入微泡造影剂Levovist后，细胞死亡率显著增加，GFP基因表达水平降低；动物实验显示，Levovist结合或不结合超声均无增强GFP基因表达水平作用，但会增加肌肉损伤面积。结论本实验条件下，Levovist无增强骨骼肌细胞基因表达作用，却加重细胞损伤。

Gene transfer with Levovist and plasmid GFP into skeletal muscle cell

Objective To explore the effect of Levovist to gene transfer in skeletal muscle cells in mice.Methods H2K myoblast cells were treated by ultrasound (1 MHz,1 W/cm~2) with pulse wave (20% duty cycle) using plasmid green fluorescent protein (GFP) in vitro.Exposure duration was 10 s,20 s,30 s,40 s,50 s and 60 s.The trend of transfection efficiency by flow cytometer and cell viability by Trypan blue assays were measured.In vivo studies,transfection efficiencies were assessed by counting GFP positive fibres on mice skeletal muscle.Tissue damage was assessed on serial sections.Results Ultrasound alone showed better trend of transfection efficiency than Levovist in vitro and addition of microbubbles had the effect to reduce cell viability significantly.In vivo,Levovist with or without ultrasound had no effect on gene transfer,instead it increased tissue damage.Conclusions(Levovist showed) no effect in plasmid DNA transfection in skeletal muscle with increased tissue damage in this experimental condition.