摘要
目的探索治疗性超声联合脂氟显微泡对人肝癌细胞HepG2基因转染的可行性及其实现高效率转染的优化参数。方法HepG2细胞交叉间隔接种于24孔板，用治疗性超声辐照含脂氟显微泡和EGFP质粒的各组细胞。分组：超声强度梯度组（A组），A1～A5亚组，分别为0．4W／cm^2、0．8w／cm^2、1．2w／cm^2、1．6W／cm^2和2．0w／cm^2；占空比梯度组（B组），B1～B3亚组，分别为10％、20％和50％；时间组（c组），C1～C3亚组，分别为30S、90s、180S。24h后用荧光显微镜观察绿色荧光蛋白表达情况，流式细胞仪检测基因转染率，台盼蓝染色检测细胞死亡率。结果不同超声强度、占空比和时间下，质粒转染效率各不相同。在一定范围内，分别增加超声强度、占空比和时间均可以提高转染效率。对于超声强度和占空比，当设置过高（声强〉1．6w／cm^2或占空比〉50％）时，由于细胞死亡率增加导致实际转染效率下降。超声强度为1．2w／cm^2时，转染率和死亡率优于A组其余各亚组；占空比为20％时，转染率和死亡率优于B组其余各亚组。当超过90S后继续增加时间，对于转染率和细胞死亡率的影响无明显统计学意义（P〉0．05）。结论超声辐照微泡是一种介导基因体外转染的有效方法，不同参数下转染率差别较大，优化参数有利于促进基因转染。
Objective To investigate the feasibility o{ transfecting gene into HepGa by therapeutic ultrasound combined with microbubble, and to explore the optimal ultrasonic parameters for high transfection efficiency. Methods HepG2 cells were cross-interval planted in 24-welI plates. EGFP plasmid DNA and microbubbles were added to the cultured HepG2 and three parameters of the therapeutic ultrasound were optimized. Firstly, set ultrasonic intensity gradient （group A, from A1 to A5）, which were 0.4 W/cm2 ,0.8 W/cm2 ,1.2 W/era2 ,1.6 W/cm2 and 2.0 W/cm2 respectively. Secondly,set the duty cycle gradient group （group B, from B1 to B3）, which were 10%, 20% and 50% respectively. Lastly, set the exposure time group（group C, from C1 to C3）, which were 30 s, 90 s and 180 s. After 24 hours, the expression of GFP was observed by fluorescence microscopy, the transfection rates was detected by flow cytometry, the cell death were assessed by trypan blue exclusion. Results The plasmid transfection efficiency was varied under different ultrasonic parameters. Within a certain range, the increasing of parameters can improve the transfection efficiency, but when the parameters were too large （eg. the intensity〉1. 6 W/cm2 or 50% duty cycle）,the actual transfection efficiency decreased due to the increased cell death. When the ultrasonic intensity was 1.2 W/cm2 , the transfection efficiency and mortality were better than the other subgroups of group A. When the duty cycle was 20 %, the transfection efficiency and mortality rate were better than the other subgroups of group B. Continue to increase the exposure time over 90 s was not statistically significant （ P 〉0.05） for transfection efficiency and cell mortality. Conclusions The ultrasound-targeted microbubble destruction is an efficient method for gene delivery in vitro. The transfection efficiency vary greatly under different parameters, so optimization parameters is conducive to the promotion of gene transfection.
出处
《中华超声影像学杂志》
CSCD
北大核心
2013年第4期344-348,共5页
Chinese Journal of Ultrasonography
基金
国家自然科学基金资助项目（81172134）
关键词
超声疗法
微气泡
转染
癌
肝细胞
Ultrasonic therapy
Microbubbles
Transfection
Carcinoma, hepatocellula
