Experimental Study In Vivo On Transferring EGFP Gene Into The Retinal Ganglion Cells Mediated By Micro Bubbles

BackgroundIt has been reported that ultrasound-mediated micro bubbles destruction could enhance obviously the transfection efficiency of EGFP gene which was transferred to cultural RGCs in vitro.ObjectivesTo validate whether ultrasound-mediated micro bubbles destruction could deliver gene EGFP to the retinal ganglion cells (RGCs) in vivo successfully, whether the transfection efficiency of this method was higher than the traditionary methods of transfecting gene and whether lead damage to RGCs.Design, time and place of the experimentObserve experiment of gene morphology. It was completed in the laboratory of Institute of Ultrasonic Image in Chongqing University of Medical Sciences between January and July in 2008.Methods 50 Sprague-Dawley rats were randomly divided into four groups:the normal control group(n=5),the"naked plasmid"group(n=15)the"plasmid with ultrasound"group (n=15 )and the"ultrasound-mediated micro bubbles"group(n=15). The normal control group: infused 5μL normal saline into the vitreous cavity of rats. The"naked plasmid"group: infused 5μL EGFP plasmids into the vitreous cavity of rats. The"plasmid with ultrasound"group: irradiated the eyes of the rats for by ultrasound immediately after infusing 5μL EGFP plasmids to the vitreous cavities. The"ultrasound-mediated micro bubbles"group: irradiated the eyes of the rats for by ultrasound immediately after infusing 5μL micro-bubbles attached with EGFP plasmids to the vitreous cavities.The acoustic intensity of the Ultrasonic Gene Transfer Therapy we used is 0.5 w/cm2,its working time was to be 1/3(that was, stopped 10s after irradiating 5s ,lasted 60s totally, the irradiating time was 20s).7 days later, harvested all the eye balls and made the retinas into whole-layer flat preparations, frozen sections and RT-PCR of EGFP mRNA。Major index of observationDetect the EGFP expression in RGCs of preparations and frozen sections by fluorescence microscope. Evaluate RGCs situation by RGCs counting.Determine the expression of EGFP mRNA by RT-PCR.Results 1.The infection efficiency of the"ultrasound-mediated micro bubbles"group was higher than the"naked plasmid"group and the"plasmid with ultrasound"group.2.No harm was detected obviously in RGCs by this method of transfecting EGFP gene by ultrasound- mediated micro bubbles.ConclusionBy the irradiation of ultrasound with low frequency, ultrasound-mediated micro bubbles destruction can deliver the reporter gene to the retinal ganglion cells effectively and securely.