Transferring EGFP Gene Into The Neovascularized Corneal Mediated By Ultrasound Microbubbles

Cornea is one of an important refracting media in ocular. Its transparency is due to its avascularity. But in the pathologic state, the new vessals growing to the transparent cornea. Corneal neovascularization is a major cause of blindness and impaired vision throughout the world. Most of people's vision loss is preventable, but only if a diagnosis is made early and appropriate therpy instituted. Then the patients quality of life would be improved. In the research field of inhibit corneal neovascular, gene theraphy becomes one of the hot spots. But gene transfection technique and gene transferction vectors is not satisfying. Thus, to find safety, efficiency, controllable and convenient gene transfection technique and gene transfection vectors is a very important task. In the long term of the ultrasounic medicine development, reseaches showed that ultrasounic medicine form diagnosis and threphy fields to the other two new application fields, that is drug delivery and gene therphy. Recent studies showed that the mechanical effect and the cavitation effect caused by ultrasound-targeted microbubbles destruction(UTMD) were able to enhance cells membrane's permeability and can increase exogenous gene into the targeted cells. This article investigate the bioeffect of ultrasound(US) microbubble(MB) on normal rabbits cornea under the different US intensity and the different exposure time. In the second part , Duplicate corneal neovascularization with suture successfully, and transfect EGFP(plamid vector coding enhanced green fluorescence protein) gene into these animal model's cornea by ultrasound microbubbles. Then find out the EGFP gene transfection rate. To investigate whether ultrasound microbubbles could directional deliver EGFP into the neovascularized cornea of rabbits and definite gene transfection efficiency. This experiment purpose is explore whether ultrasound microbubbles is a new treatment for corneal neovasclar disease as a targeted carrier.This reaseach include two sections:PARTⅠThe bioeffect of ultrasound microbubble on corneal with ultrasound exposureObjective To investigate the bioeffect of US microbubbles on rabbit cornea in different US intensity and exposure time. Methods The cornea were exposed to US under different parameter (US Intensity: 0.5, 1.0, 2.0 W/cm~2; US Exposure time: 30, 60, 120 seconds), and then observed by corneal quantitative analysis and histological assessment . Results There was statistical significance among 0.5,1.0, 2.0 W/cm~2 in endothelial cell density and percentage of hexagonal(p<0.05). Compare to the other two groups, there was statistical significance in the 120s US exposure time(p<0.05). Conclusions Ultrasound microbubbles lower the threshold for cavitation by ultrasound energy.Limited ultrasound energy has great influence on cornea.PARTⅡTransferring EGFP gene into the Neovascularized Corneal Mediated by Ultrasound MicrobubblesObjective To explore whether ultrasound microbubbles could directional deliver EGFP into the neovascularized cornea of rabbit and definite gene transfect efficiency. Method After use suture induced corneal angiogenesis, New Zealand albino rabbits were randomly divided into four groups: Group A were the naked plasmid DNA of EGFP into rabbit subconjunctival; Group B were taken ultrasound irradiation after injected EGFP immediately; The plasmid DNA of EGFP and microbubbles complex were injected into rabbit subconjunctival in group C; Group D were injectioned the complex(plasmid+MB) and then take US immediately. The results were observed by histopathologic and Leica confocal microscope. Results Group D greatly enhance plasmid DNA of EGFP expressed in cornea. The ratio of EGFP positive was 133.03±21.99. And the three other group were 75.17±7.08; 97.02±8.31 and 81.83±9.44 respectively. There have statistical significance(F=23.56 , P<0.05). Conclusions Limited ultrasound energy could obviously improve plasmid DNA of EGFP transfect efficiency in neovascularized corneal of rabbits.