超声造影剂Optison介导质粒GFP转染小鼠骨骼肌细胞的实验研究

目的 探讨微泡造影剂Optison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(1 MHz脉冲波,20%工作周期,空间时间峰值强度1 W/cm2)结合Optison作用于小鼠体外H2K成肌细胞,照射时间分别为10、20、30、40、50及60 s,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Optison作用于小鼠胫前肌,1周后处死小鼠,荧光显微镜检测GFP阳性肌纤维数,HE染色后计算肌肉损伤面积.结果 活体外细胞实验结果显示,与阳性对照组相比,Optison结合超声作用于H2K细胞10、20及30 s时,显著增强GFP基因表达水平(P＜0.01),但于40、50及60 s时基因表达水平显著降低(P＜0.01),细胞死亡率总体显著增加(P＜0.01).动物实验结果显示,Optison单独或结合超声均显著增强GFP基因表达水平,且Optison单独作用显著减少肌肉损伤面积.结论 Optison可显著增强活体小鼠骨骼肌细胞基因表达水平,同时具有肌肉保护作用.

Ultrasound and contrast Optison induced gene plasmid GFP transfer into skeletal muscle cell

Objective To explore the effect of Optison on gene transfer into skeletal muscle cells in mice. Methods H2K myoblast cells were treated by Optison and ultrasound (1 MHz, 1 W/cm2) with pulsed wave (20% duty cycle) using plasmid GFP (Green Fluorescent Protein) 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 area was assessed on serial sections. Results GFP transfection efficiency at 10 s, 20 s, 30 s was enhanced and reduced at 40 s, 50 s, 60 s significantly for H2K cell treated by Optison + ultrasound compared with positive control in vitro with significantly increased cell killing rate. In vivo, Optison with or without ultrasound had significant effect on gene transfer and Optison alone showed significantly reducing tissue damage. Conclusions Optison could enhance gene transfection efficiency and protect skeletal muscle fibres in mice in vivo.