治疗性超声介导微泡破裂促进大鼠体内肌肉基因转染的参数优化实验研究

Study on the Parameter Optimization for Therapeutic Ultrasound Mediated Microbubble Destruction Enhancs Gene Transfection in Rat Muscle in vivo

目的探讨治疗性超声介导微泡破裂促进基因体内转染大鼠肌肉的最适合的转染条件。方法在大鼠双侧胫前肌内局部注射微泡与增强型绿色荧光蛋白(EGFP)质粒混合物,应用不同输出功率、不同占空比及不同超声辐照时间的治疗性超声辐照胫前肌,分别用肌肉局部注射及静脉注射微泡和质粒联合超声辐照,根据荧光染色及免疫组化染色下EGFP表达结果判断转染效果,根据转染效果最好且HE染色下肌肉损伤最小选出最适合的超声辐照条件和最适合的注射方式。将大鼠分为4组:①超声辐照+微泡+质粒组,②微泡+质粒组,③超声辐照+质粒组,④单纯质粒组。选取最适辐照及注射方式后,将大鼠在超声辐照后5d处死,荧光染色及免疫组化染色下观察肌肉EGFP表达情况,HE染色观察肌肉损伤情况。结果在1MHz治疗性超声辐照下,输出功率2 W/cm2,占空比20%,超声辐照3min的最适合的辐照条件下,肌肉EGFP表达明显,且无明显损伤。肌肉局部注射较静脉注射更适合。荧光染色和免疫组化染色示4组中超声辐照+微泡+质粒组肌肉EGFP表达高于其余3组,微泡+质粒组高于其余2组(P<0.05)。HE染色下未见超声辐照及微泡造成的肌肉损伤。结论在适合转染条件下,治疗性超声联合微泡能明显增强基因体内转染大鼠肌肉的效率,且不损伤肌肉细胞,可作为基因治疗的一种安全有效的非病毒性基因转染方法。

Objective To search for the most suitable gene transfection conditions for rat muscle in vivo by therapertic ultrasound-mediated microhubble destruction （UMMD）. Methods A mixture of microbubhles and enhanced green flurescence protein （EGFP） plasmids was injected into rat tibialis anterior muscle and the muscels treated with ultrasound irradiation by different output intensity, duty cycle and irradiation time of therapeutic ultrasound. The transfection efficiency was demonstrated by the EGFP expression results under fluorescent staining and immunohistochemical staining. And the most favorable ultrasound conditions as well as local intra-muscle and intravenous injection methods were selected based on the best transfection efficiency and the least muscle damage under HE staining. The rats were divided into four groups： （D ultrasound d- microbubbles ^- plasmid; （2） microbubble -t- plasmid; （~） ultrasound -/- plasmid; @ plasmid only. The favorable ultrasound conditions and injection method were selected out on the basis of above steps. EGFP expression was observed in the tibialis anterior muscle of each group. The rats were sacrificed in groups at 5 days after they underwent ultrasound irradiation, the EGFP expression in muscle was observed after fluorescent and immunohistochemical stainingand the muscle damage was also observed under HE staining. Results The most favorable conditions consisted of a 1-MHz therapeutic ultrasound irradiation applied for 3 min, a power output of 2 W/cm2 and a 20% duty cycle. Under these conditions, the muscle showed significant EGFP expression, and the muscle was not significantly damaged. The EGFP expression induced by the local intra-muscle injection was more significantly increased than that induced by the intravenous injection. Among the four groups, the EGFP expression under fluorescence staining and immunohistochemical staining in the muscle of the ultrasound d- microbuhbles ~ plasmid group was significantly higher than that of the other three groups, and the microbubbles -＋- plasmid group was higer than that of the other two groups （P〈0. 05）. No muscle damage caused by the ultrasound and microbubbles was detected under HE staining. Conclusion Under the optimal transfection conditions, the therapeutic ultrasound-mediated microbubble destruction method can significantly enhance the in vivo gene transfection efficiency of rat muscle and found no damage to the muscle. Thus, these conditions can be used as part of a safe and effective non-viral gene transfection procedure for gene therapy.