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

<正>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 2-4μm 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 5μL normal saline to the vitreous cavity;the naked plasmid group （n=15） was infused with 5μL 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.