不同转染方式介导基因转染效果的评价

Evaluation of transfect efficiency in different methods

目的 评价不同转染方式介导基因转染的效果.方法 将增强型绿色荧光蛋白（eGFP）基因转染至293T细胞中.根据转染方式不同分为6组：对照组、单纯质粒组（eGFP）、微泡＋质粒组（MB＋eGFP）、辐照＋质粒组（US＋ eGFP）、超声辐照＋微泡＋质粒组（UTMD＋ eGFP）、脂质体＋质粒组（Lip2000＋eGFP）.超声辐照条件为2.0 W/cm2、45 s、1 MHz、20％占空比,eGFP质粒浓度为1 mg/ml.将293T细胞种植于24孔板内,细胞密度1.5×10^5/孔,24 h后进行转染.转染72 h荧光显微镜下观察细胞内绿色荧光蛋白的表达,流式细胞仪检测荧光细胞比例,CCK-8检测细胞存活率,计算转染效率（=荧光细胞比例×细胞存活率）;westem bloting检测eGFP蛋白表达水平.结果 转染后72 h,UTMD＋ eGFP组和Lip2000＋eGFP组荧光细胞表达较多.各组的转染效率分别为：0％（对照组）、0％（eGFP组）、0％（MB＋ eGFP组）、1.9％（US＋ eGFP组）、33.8％（UTMD＋ eGFP组）、74.8％（Lip2000＋ eGFP组）.Western显示eGFP在UTMD＋ eGFP组和Lip2000＋ eGFP组显著表达,其余各组无明显表达.结论 超声靶向微泡破灭和脂质体转染均能有效促进基因转染.

Objective To investigate the transfection efficiency from different methods.Methods The green fluorescent protein gene was transfected into 293T cells.Different transfection methods were applied to six groups of cells.Group one：no transfection; group two：only eGFP; group three：microbubbles ＋ eGFP;group four：ultrasound exposure ＋ eGFP;group five：ultrasound-targeted microbubbles destruction ＋ eGFP;group six：Liposome 2000 ＋ eGFP.The parameters of ultrasound oscillation were 2.0 W/cm2,45 s,1 MHz,20% duty cycle（DC）,the eGFP DNA concentration was 1mg/ml.293T cells were cultured in 24-well plates for 24 hours ahead of transfection study.72 hours later,fluorescence microscope was used to observe the green fluorescent protein in 293T cells,flow cytometer was used to calculate the cells with green fluorescent proteins expressing,and the viability of 293T cells was assessed by CCK-8,the efficiency of transfection was calculated from the viability multipled by the proportion of 293T cells expressing green fluorescent protein.Western blot （WB） was performed to differentiate the expression levels of GFP in each group.Results 72 hours post transfection,both group five and group six showed many green fluorescent cells under fluorescence microscope and high expression of GFP which was displayed by WB.The transfection efficiency of each group was 0%,0%,0%,1.9%,33.8%,74.8%,respectively.Conclusions Both ultrasound-targeted microbubbles destruction and liposome can enhance the gene transfection efficiently.