Tang Yuan-Jiao, Zhang Ling-Yan, Wang Lei, Lin Ling, Wen Xiao-Rong, Qiu Li
Department of Ultrasound, West China Hospital, Sichuan University, Chengdu 610041, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2012 Nov;43(6):930-5.
To search for the most suitable gene transfection conditions for rat muscle in vivo by therapertic ultrasound-mediated microbubble destruction (UMMD).
A mixture of microbubbles 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: 1) ultrasound + microbubbles + plasmid; 2) microbubble + plasmid; 3) ultrasound + plasmid; 4) 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.
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 + microbubbles + 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.
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.
通过治疗性超声介导的微泡破坏（UMMD）寻找大鼠体内肌肉最合适的基因转染条件。
将微泡与增强型绿色荧光蛋白（EGFP）质粒的混合物注入大鼠胫前肌，然后用不同输出强度、占空比和照射时间的治疗性超声对肌肉进行照射。通过荧光染色和免疫组化染色下的EGFP表达结果来证明转染效率。并根据最佳转染效率和苏木精-伊红（HE）染色下最小的肌肉损伤，选择最有利的超声条件以及局部肌肉内和静脉注射方法。将大鼠分为四组：1）超声+微泡+质粒；2）微泡+质粒；3）超声+质粒；4）仅质粒。根据上述步骤选出有利的超声条件和注射方法。观察每组大鼠胫前肌中EGFP的表达情况。在超声照射后5天对大鼠进行分组处死，荧光和免疫组化染色后观察肌肉中EGFP的表达情况，同时在HE染色下观察肌肉损伤情况。
最有利的条件包括应用1MHz治疗性超声照射3分钟，功率输出为2W/cm²，占空比为20%。在这些条件下，肌肉显示出显著的EGFP表达，且肌肉未受到明显损伤。局部肌肉内注射诱导的EGFP表达比静脉注射诱导的显著增加。在四组中，超声+微泡+质粒组肌肉的荧光染色和免疫组化染色下的EGFP表达明显高于其他三组，微泡+质粒组高于其他两组（P<0.05）。在HE染色下未检测到超声和微泡引起的肌肉损伤。
在最佳转染条件下，治疗性超声介导的微泡破坏方法可显著提高大鼠肌肉的体内基因转染效率，且未发现对肌肉有损伤。因此，这些条件可作为基因治疗安全有效的非病毒基因转染程序的一部分。
