低频超声照射SonoVue对绿色荧光蛋白质粒在小鼠肝癌移植瘤中表达的作用

Effect of microbubble contrast agent on expression of plasmid EGFP in hepatocellular carcinoma of mice with exposure to low-frequency ultrasound

目的 研究物理参数对体内基因转染率的影响，确定最佳剂量-效应关系。方法建立小鼠（C57BL／6J）肝癌皮下移植瘤模型，经尾静脉注入绿色荧光蛋白质粒（pEGFP）和SonoVue混合物，给予不同辐照声强（1W／cm^2、2w／cm^2、3w／cm^2）、辐照时间（1min、5min、10min）以及不同剂量SonoVue（30μl、60μl、90μl）。流式细胞仪和荧光显微镜检测肿瘤细胞内绿色荧光蛋白表达。结果pEGFP在肿瘤组织内的表达随辐照时间、辐照声强、造影剂剂量的增加而增多，持续增加辐照时间、声强和造影剂剂量并不能有效增加其转染率，2W／cm^2[（21．02±1．45）％]、5min[（23．22±1．91）％]、60plSonoVue[（21．02±1．45）％]时，pEGFP在肿瘤组织内的表达达到最大。结论不同物理参数对体内基因转染率有明显影响，2W／cm^2、5min、60μlSonoVue的参数条件下，可达到最佳剂量-效应关系。

Objective To explore the optimal dose-effect relationship of gene transfer according to the expression of pEGFP in tumor cells under different parameters mediated by microbubble contrast agent and ultrasound in vivo. Methods C57BL/6J mice were inoculated subcutaneously in the middle of the right flank with hepa1- 6 tumor cells. The mixture of SonoVue and enhanced green fluorescent protein（EGFP） plasmid was injected into the tail vein of each mouse, groups were randomly established according to different output intensity（ 1 W/cm^2 , 2 W/cm^2 ,3 W/cm^2 ）, exposure time （ 1 min, 5 min, 10 min） and volume of SonoVue（30μl,60 μl,90μl）, the expression of EGFP in tumor cells under different parameter was examined by flow cytometry and fluorescence microscopy. Results The expression of pEGFP in tumor cells was significantly higher with the increase of exposure time,output intensity and volume of SonoVue （1 min vs 5 min, P 〈0.05 ; 1 W/cm^2 vs 2 W/cm^2 , P 〈0. 05 ; 30μl vs 60 μl, P 〈0. 05）. However, the transfection efficiency of EGFP can not be enhanced continuously with the increasing exposure time,output intensity and volume of SonoVue （5 min vs 10 rain, P〉0. 05; 2 W/cm^2 vs 3 W/cm^2 , P 〉0.05, 60 μl vs 90 μl, P 0.05）. The highest transfection efficiency was achieved under intensities was 2 W/cm^2 [-（21.02±1.45）%] with 5 minE（23.22±1.91 ）%] exposed to ultrasound, and 60 μl SonoVue [（21.02±1.45）%]. Conclusions Gene transfection efficiency changded with the difference parameters. 2 W/cm^2 ,5 min and 60 μl SonoVue led to the optimum dose-effect relationship of gene transfer in vivo.