超声联合微泡介导缺氧诱导因子-1α基因转染293T细胞的实验研究

Experimental Study of Hypoxia Inducible Factor-1 Aplha Transfected into 293T Cells by Ultrasound-Mediated Microbubble Destruction

目的:探讨超声联合微泡造影剂介导缺氧诱导因子(HIF)-1α基因转染人胚肾293T细胞的转染效率。方法:将6孔板中的293T细胞悬液分为3组,A组:质粒+超声辐照组,加入质粒,终浓度为15μg/孔;B组:载基因微泡+超声辐照组,加入载基因质粒微泡混合液,质粒的终浓度为15μg/孔,微泡的终浓度为200μl/孔;C组:对照组,每孔中仅加入单纯质粒DNA,终浓度15μg/孔。A组和B组均接受超声辐照,超声照射条件为连续波,声强1.5 W/cm2,辐照时间30s。超声辐照转染48h后,用荧光显微镜和流式细胞仪分别定性定量观察各组细胞基因的转染效率。结果:转染48h后,荧光显微镜观察到转染成功的细胞内发出绿色荧光,流式细胞仪检测A组、B组、C组的基因转染效率分别为(5.6±0.5)%、(30.5±1.0)%、(0.1±0.1)%。结论:超声辐照联合微泡能够介导治疗性基因的体外细胞转染,为冠心病心肌缺血的HIF-1α基因治疗奠定基础。

Objective：To investigate the transfection efficiency of hypoxia inducible factor（HIF）-1αin the 293 Tcells mediated by ultrasound microbubble contrast media.Methods：293Tcells suspension of six orifice plates were divided into three groups.Group A,the plasmid-ultrasound group,was given 15μg plasmid only.Group B,the plasmid-microbubbles-ultrasound group,was given15μg HIF-1αplasmid and 200μg microbubbles.Group C,the contrast group,was given 15μg HIF-1αplasmid only.Group A and group B were exposed to ultrasound（1.5W/cm2）for 30 s.48hours later,the transient expression rate was observed by fluorescence microscopy and flow cytometry.Results：Green fluorescence was observed in group A,B and C by fluorescence microscopy.The transfection efficiency was（5.6±0.5）%in the group A,（30.5±1.0）%in the group B,and（0.1±0.1）%in the group C,respectively.Conclusion：Ultrasound-mediated microbub-bles destruction can encourage gene transfection in cells.