超声微泡介导增强型绿色荧光蛋白基因转染视网膜神经节细胞的体内实验

BACKGROUND： Studies have demonstrated that ultrasound-mediated microbubble destruction significantly improves transfection efficiency of enhanced green fluorescent protein （EGFP） in in vitro cultured retinal ganglial cells （RGCs）. OBJECTIVE： To investigate the feasibility of ultrasound-mediated microbubble destruction for EGFP transfection in rat RGCs, and to compare efficiency and cell damage with traditional transfection methods. DESIGN, TIME AND SETTING： In vivo, gene engineering experiment. The study was performed at the Central Laboratory, Institute of Ultrasonic Imaging, Chongqing Medical University from March to July 2008. MATERIALS： Eukaryotic expression vector plasmid EGFP and microbubbles were prepared by the Institute of Ultrasonic Imaging, Chongqing Medical University. The microbubbles were produced at a concentration of 8.7 × 10^11/L, with a 2-4 μm diameter, and 10-hour half-life in vitro. METHODS： A total of 50 Sprague Dawley rats were randomly assigned to four groups. Normal controls （n = 5） were infused with 5 μL normal saline to the vitreous cavity; the naked plasmid group （n = 15） was infused with 5 pL EGFP plasmid to the vitreous cavity; in the plasmid with ultrasound group （n = 15）, the eyes were irradiated with low-energy ultrasound wave （0.5 W/cm^2） for a total of 60 seconds （irradiated for 5 seconds, at 10-second intervals） immediately following infusion of EGFP plasmids to the vitreous cavities. In the microbubble-ultrasound group （n = 15）, the eyes were irradiated with the same power of ultrasonic wave immediately following infusion of microbubbles containing EGFP plasmids to the vitreous cavities. MAIN OUTCOME MEASURES： After 7 days, retinal preparations and EGFP expression in RGCs were observed by fluorescence microscopy. RGC quantification in the retinal ganglion cell layer was performed. In addition, EGFP mRNA expression was semi-quantitatively determined by RT-PCR. RESULTS： The transfection efficiency of EGFP to RGCs by microbubbles with ultrasound was significantly greater than the other groups, and no obvious damage was detected in the RGCs. CONCLUSION： Under irradiation of low-frequency ultrasound waves, ultrasound-mediated microbubble destruction was effective and resulted in safe transfection of the EGFP gene to the RGCs.

In vivo transfection of enhanced green fluorescent protein in rat retinal ganglion cells mediated by ultrasound-induced microbubbles

BACKGROUND:Studies have demonstrated that ultrasound-mediated microbubble destruction significantly improves transfection efficiency of enhanced green fluorescent protein (EGFP) in in vitro cultured retinal ganglial cells (RGCs).OBJECTIVE:To investigate the feasibility of ultrasound-mediated microbubble destruction for EGFP transfection in rat RGCs,and to compare efficiency and cell damage with traditional transfection methods.DESIGN,TIME AND SETTING:In vivo,gene engineering experiment.The study was performed at the Central Laboratory,Institute of Ultrasonic Imaging,Chongqing Medical University from March to July 2008.MATERIALS:Eukaryotic expression vector plasmid EGFP and microbubbles were prepared by the Institute of Ultrasonic Imaging,Chongqing Medical University.The microbubbles were produced at a concentration of 8.7×1011/L,with a 2-4 μm diameter,and 10-hour half-life in vitro.METHODS:A total of 50 Sprague Dawley rats were randomly assigned to four groups.Normal controls (n=5) were infused with 5 μL normal saline to the vitreous cavity;the naked plasmid group (n=15) was infused with 5 μL EGFP plasmid to the vitreous cavity;in the plasmid with ultrasound group (n=15),the eyes were irradiated with low-energy ultrasound wave (0.5 W/cm2) for a total of 60 seconds (irradiated for 5 seconds,at 10-second intervals) immediately following infusion of EGFP plasmids to the vitreous cavities.In the microbubble-ultrasound group (n=15),the eyes were irradiated with the same power of ultrasonic wave immediately following infusion of microbubbles containing EGFP plasmids to the vitreous cavities.MAIN OUTCOME MEASURES:After 7 days,retinal preparations and EGFP expression in RGCs were observed by fluorescence microscopy.RGC quantification in the retinal ganglion cell layer was performed.In addition,EGFP mRNA expression was semi-quantitatively determined by RT-PCR.RESULTS:The transfection efficiency of EGFP to RGCs by microbubbles with ultrasound was significantly greater than the other groups,and no obvious damage was detected in the RGCs.CONCLUSION:Under irradiation of low-frequency ultrasound waves,ultrasound-mediated microbubble destruction was effective and resulted in safe transfection of the EGFP gene to the RGCs.