低频超声联合微泡造影剂促进增强型绿色荧光蛋白质粒转染裸鼠人前列腺癌皮下移植瘤的实验研究

目的 探讨低频超声联合微泡造影剂促进增强型绿色荧光蛋白质粒（pEGFP）转染裸鼠人前列腺癌皮下移植瘤的可行性，并观察不同超声声强对转染率的影响。方法 BABL/c裸鼠30只建立人前列腺癌皮下移植瘤模型，然后随机分为单纯低频超声组和低频超声＋微泡组2组，每组各15只。单纯低频超声组裸鼠分别经尾静脉注射质粒与0.9%氯化钠混悬液250μl（其中含pEGFP质粒50μg），低频超声＋微泡组裸鼠分别经尾静脉注射质粒与微泡混悬液250μl（其中含pEGFP质粒50μg）。每组裸鼠再分为3个亚组，每组各5只，分别以声强为1、2、3 W/cm^2的超声（频率80 kHz，占空比50%）辐照10 min。超声辐照后第3天采用激光共聚焦显微镜观察各组绿色荧光蛋白表达情况并分析各组平均荧光强度，同时进行常规病理学检查。采用单因素方差分析比较单纯低频超声组及低频超声＋微泡组平均荧光强度，进一步组间两两比较采用SNQ-q检验。结果 1、2、3 W/cm^2低频超声＋微泡组平均荧光强度分别为23.75±2.54、30.25±1.67、59.60±2.03，均高于相对应的1、2、3 W/cm^2单纯低频超声组平均荧光强度14.04±1.35、14.66±0.98、14.32±1.20，且差异均有统计学意义（q值分别为18.26、14.12、13.88，P均＜0.05）；且随着超声声强的增加，低频超声＋微泡组平均荧光强度也增高，且差异均有统计学意义（q值分别为15.33、17.81、13.79，P均＜0.05），说明随着超声声强的增强，低频超声＋微泡组转染率明显增高。单纯低频超声组均未见明显的绿色荧光。常规病理学检查显示各组均未对组织造成明显的损伤。结论 超声联合微泡造影剂能有效促进pEGFP质粒转染裸鼠人前列腺癌皮下移植瘤，而在一定范围内增高声强能提高转染率。

Experimental study of low-frequency ultrasound combined with microbubbles promote enhanced green fluorescent protein plasmid transfecting into transplanted prostate cancer of nude mice

Objective To investigate the feasibility of low-frequency ultrasound combined with microbubbles improving transfection of enhanced green lfuorescent protein plasmid （pEGFP） to subcutaneously transplanted prostate cancer in nude mice, and to optimize the parameters of intensity. Methods The model of nude mice of subcutaneously transplanted prostate cancer were built. Then they were divided into 2 groups including low-frequency ultrasound group and low-frequency ultrasound combined with microbubbles group, each group with 15 mice. Then 250μl of mixture of saline and pEGFP （50μg） were co-injected in low-frequency ultrasound group and 250μl of mixture of SonVue and pEGFP （50μg） were co-injected in low-frequency ultrasound combined with microbubbles group through tail vein. According to intensity, they were divided into 3 subgroups respectively, including group 1 W/cm^2, group 2 W/cm^2 and group 3 W/cm^2, each group with 5 mice.＆amp;nbsp;Ultrasound was applied at 80 kHz input frequency with 50%duty cycle for 10 minutes after pEGFP injection. The tumors were collected on the third day after treatment. The expression of pEGFP in tumor was examined by laser scanning confocal microscope （ISCM） and the average lfuorescence intensity were estimated. At the same time, routine pathological examination was performed. The mean lfuorescence intensity of low frequency ultrasound group and low frequency ultrasound combined with microbubble group were compared with one-way ANOVA and those of any two groups were compared by SNQ-q test. Results TThe mean lfuorescent light in low-frequency ultrasound combined with microbubbles group were 23.75±2.54, 30.25±1.67 and 59.60±2.03, which were obviously stronger than those of low-frequency ultrasound treatment group （14.04±1.35, 14.66±0.98 and 14.32±1.20）, the difference was statistically signiifcant （the value of q were 18.26, 14.12 and 13.88, P〈0.05）. The rate of gene transfection increased along with the enhancement of the ultrasound intensity （the value of q were 15.33, 17.81 and 13.79, P＆〈0.05）. Histology analyses performed by HE staining showed that there was no damage to the tumor tissues in all groups, tumor tissues were intact and without infection. Conclusions Low-frequency ultrasound combination with microbubbles can significantly enhance pEGFP transfecting into subcutaneously transplanted prostate cancer in nude mice. In certain range, improving the ultrasound intensity can increase the rate of gene transfection.