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

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10 μg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10 μg/孔,微泡200 μl/孔;C组,脂质体+质粒组,每孔加入4 μl脂质体和5 μg质粒;D组,对照组,每孔仅加入10 μg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1.5 W/cm2,作用时间为30 s.转染后48 h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 48 h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.

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

Objective To investigate the transfection efficiency of Ang-1 in the 293T cells mediated by ultrasound-microbubble contrast media. Methods 293T cells suspension of six orifice plates were divided into four groups: The plasmid-ultrasound group(A group) was given 10μg Ang-1 plasmid only. The plasmid-microbubbles-ultrasound group(B group) was given 10μg Ang-1 plasmid and 200μg microbubbles. The plasmid-liposome group(C group) was given 4μg liposome and 5μg 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 ultrasound (1.5 W/cm2) 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 microscopy. 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.