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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/cm2)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。
Objective To investigate the effect of air-based microbubble contrast agent Levovist-mediated gene transfer in mouse skeletal muscle cells. Methods Plasmid GFP was used as the target gene and treated with Levovist in H2K myoblasts by ultrasound (1MHz pulse wave, 20% duty cycle and peak intensity 1W / cm2 in space time) for 10s, 20s, 30s, 40s, 50s, 60s, GFP-positive cells by flow cytometry, cell survival rate by trypan blue staining. Ultrasound combined with Levovist acting on the anterior tibial muscle of mice, mice were sacrificed one week later, the number of GFP positive myofibers was measured by fluorescence microscopy, and the area of muscle destruction was estimated by HE staining. Results Ultrasound treatment of H2K cells alone showed better effect of enhancing GFP gene expression. The addition of microbubble contrast agent Levovist significantly increased the cell death rate and decreased the expression of GFP gene. Animal experiments showed that Levovist with or without ultrasound No enhancement of GFP gene expression levels, but will increase the muscle damage area. Conclusion Under the experimental conditions, Levovist did not enhance the gene expression of skeletal muscle cells, but increased cell damage.