超声辐照联合微泡造影剂介导人血管生成素-1基因体外转染293T细胞的研究

Experimental study of hAng-1 transfected into 293T cells by ultrasound-mediated microbubble destruction

目的探讨超声辐照联合微泡造影剂的基因转染系统介导人血管生成素-1（hAng-1）基因转染人胚肾293T细胞的转染效率。方法将六孔板中的293T细胞悬液分为4组：A组，超声＋质粒组，每孔加入10μg目的基因质粒；B组，超声＋微泡＋质粒组，每孔加入微泡和质粒混合液，终浓度分别为质粒10pg／孔，微泡200μL／孔；C组，脂质体＋质粒组，每孔加入4μl脂质体和5μg质粒；D组，对照组，每孔仅加入10μg目的基因质粒。A组和B组均接受超声辐照，照射条件为连续波，声强1．5W／cm^2，作用时间为30s。转染后48h用荧光显微镜和流式细胞仪分别定性、定量观察基因的转染效率。结果48h后，荧光显微镜下观察到转染成功的细胞胞浆内发出绿色荧光，流式细胞仪检测A组、B组、C组、D组的基因转染效率分别为（3．5±0．4）％、（20．8±1．2）％、（18．0±0．9）％和（0．2±0．1）％。结论超声本身即有促进基因转染的作用，超声辐照联合微泡造影剂后则增强了这种作用，明显增加了基因的转染效率。

Objective To investigate the transfection efficiency of Ang-1 in the 293T cells mediated by ultrasoundmicrobubble contrast media. Methods 293T cells suspension of six orifice plates were divided into four groups： The plasmidultrasound group（A group） was given 10μg Ang-1 plasmid only. The plasmid-mierobubbles-uhrasound group（ B group） was given 10μg Ang-1 plasmid and 200μg microbubbles. The plasmid-liposome group （C group ） was given 4μg liposome and 5 μ g Ang-1 plasmid. The contrast group （D group） was given 10μg Ang-1 plasmid only. A group ,B group and C group were exposed to uhrasound （1.5 W/cm^2） for 30s. Forty-eight hours later, the transient expression rate was observed by fluorescence microscopy and flow cytometry. Results Green fluorescence was observed in A, B, C and D group by fluorescence micrdseopy. The transfection efficiency was （3.5 % ±0.4 ）% in the A group, （20.8% ± 1.2 ）% in the B group, （18.0% ± 0.9）% in the C group and （0.2% ± 0. 1 ）% in the D group, respectively. Conclusion Ultrasound can encourage gene transfection in cells, whereas microbubbles combined with ultrasound exposure can improve the transfection efficiency obviously.