超声微泡破裂法促进骨形态发生蛋白-2在小鼠骨骼肌表达的研究

目的探讨超声微泡破裂法对基因重组人骨形态发生蛋白-2（rhBMP-2）质粒pIRES-rhBMP2-EGFP在小鼠骨骼肌的转染、表达的促进作用。方法24只BALB/c小鼠随机分为4组，每组6只。A组和B组选择右侧胫前肌为注射点，其中A组注射质粒，B组注射质粒与微泡造影剂混合物后用超声辐照，质粒注射量为30 μg；C组和D组选择右侧股四头肌为注射点，其中C组注射质粒，D组注射质粒和微泡造影剂混合物后用超声辐照，质粒注射量为100 μg。7 d后处死A组和B组小鼠，取胫前肌在荧光显微镜下观察绿色荧光蛋白的表达情况；14 d后处死C组和D组小鼠，取股四头肌行免疫组化检测rhBMP-2表达情况。结果7 d后B组阳性肌纤维百分率高于A组（P<0.05）；14 d 后，C组和D组都可检测到rhBMP-2的表达，但D组rhBMP-2表达量比C组多。结论超声介导微泡破裂法能增加外源性rhBMP-2基因在小鼠体内骨骼肌转染率和表达水平，有望为牙周病的基因治疗提供一种新型方法。

Ultrasound-mediated microbubble destruction enhances bone morphogenetic protein-2 gene expression in mouse skeletal muscles

Objective To explore whether ultrasound-mediated microbubble destruction can enhance the expression efficiency of plasmid pIRES -rhBMP2 -EGFP for bone morphogenetic protein -2（BMP -2） in mice skeletal muscle. Methods Twenty four male BALB/c mice were divided into four groups. The naked plasmid was injected into the pretibial muscle or the quadriceps muscle（group A and group C） without ultrasound-mediated microbubble destruction method. Micobubbles with plasmid were injected into the pretibial muscle or the quadriceps muscle（group B and group D） with destructing microbubbles by ultrasound immediately. Twelve mice（group A and group B, 30 μg plasmid  injected） were killed after 7 days and the tissue samples of the pretibial muscle were obtained to observe the expression of enhanced green fluorescence protein（EGFP） by inverted fluorescence microscope, gene transfection efficiencies were quantified by counting EGFP positive fibers on mice skeletal muscle. After 14 days, the other twelve mice（group C and group D, 100 μg plasmid injected） were killed and immunnohistochemical technique was applied to detect the rhBMP-2 gene expression. Results The percentage of GFP-positive fibers was significantly lower in the group A than that in the group B. After 14 days, expression of rhBMP-2 was detected in cells and interstitial spaces in the group C and group D, and expression efficiency of rhBMP-2 in the group D was significantly higher than that in the group C. Conclusion Ultrasound-mediated microbubble destruction could enhance the transfection and expression efficiency of rhBMP-2 gene in skeletal muscle of mouse in vivo. It is a new gene therapy method for periodontal regeneration.