超声和微泡造影剂介导细胞基因转染的实验研究  

Ultrasound and microbubbles:their functions in gene transfer in vitro

作　　者：陈云超[1] 张青萍[1] LIANG Hai-dong 朱蔚[1] 张超[1] Martin JK Blomley LU Qi-long 

机构地区：[1]华中科技大学同济医学院附属同济医院超声影像科,武汉430030 [2]Department of Imaging,Clinical Sciences Centre, Imperial College, Hammersmith Hospital, London, UK

出　　处：《中华超声影像学杂志》2006年第11期864-868,共5页Chinese Journal of Ultrasonography

基　　金：国家留学回国人员科研启动基金(教外司留[2005]383号);国家自然科学基金项目(30670620)

摘　　要：目的 探讨低频超声对细胞基因转染的作用。方法 超声治疗仪频率1MHz，脉冲重复频率100Hz，占空系数20％。质粒DNA为含编码绿荧光蛋白的pEGFP。应用荧光显微镜和流式细胞仪评价细胞基因转染率，台盼蓝染色计算细胞成活率。选用C2C12、3T3-MDEI和CHO3种细胞系为研究对象，加入DNA后辐照不同声强、时间或加入超声造影剂，观察各条件下的细胞基因转染率和成活率。结果 ①超声介导的基因转染与声强和辐射时间有关，最佳剂量为1w／cm^2 20s；②同样超声剂量，较高的声强较早达到最大基因转染率；③较低剂量时，微泡造影剂可使超声介导的基因转染提高2～3倍并可显著提高最高基因转染率。结论 低频超声可介导细胞基因转染，基因转染率不但与超声辐射剂量有关，而且同样剂量时，高声强较早达到最大基因转染率，最佳剂量是1w／cm^2 20s。同时，微泡造影剂可提高超声介导基因转染的最高转染率。Objective To observe the functions of low-frequency ultrasound system in gene delivery in vitro. Methods The frequency of the ultrasound system was 1MHz and the pulse-repetition frequency was 100 MHz with fixed 20% duty cycle. Plasmid encoding green fluorescent protein（pEGFP） was used as a reporter. Transfection rates and cell viability were assessed by fluorescent microscopy, flow cytometry and trypan blue exclusion. The cells was exposed with varying ultrasound intensities and duration to evaluate their effects on cell viability and transfection efficiency in three cell lines （C2C12,3T3-MDEI and CHO）. Microbubble, Optison（10 %）, was added to observe its function in gene transfeetion. Experiments were performed in triplicate. Results （1）As the ultrasound intensity and the exposure time increasing, the cell transfeetion rate increased and the cell viability decreased, but at high energy intensities, the cell viability decreased dramatically which induced the cell transfeetion rate decrease. The most efficiency ultrasound intensity to induce gene transfer was 1 W/cm^2 with duration of 20s. （2） In the same energy intensity, different ultrasound intensity induced different cell gene transfer rate and viability. Higher ultrasound intensity met its maximum gene transfer rate first and had higher cell toxicity as well. （3） Mierobubble could increase ultrasound induced cell gene transfer rate about 2 to 3 times mainly in lower energy intensities. Moreover, mierobubble could raise the maximum gene transfer rate mediated by ultrasound. Conclusions Therapeutic ultrasound system can induce cell gene transfer. The cell gene transfer rate and viability relate not only the ultrasound energy intensity but also the ultrasound intensity, which the higher ultrasound intensity achieve its maximum transfer rate first and the most efficiency ultrasound intensity to induce gene transfer is 1 W/cm^2 with duration of 20 s, whilst the mierobubbles can significantly increase its maximum gene transfer rate in vitro.

关 键 词：超声检查 造影剂 基因转移技术 

分 类 号：R346[医药卫生—基础医学]