Study On Ultrasound-Targeted Microbubble Destruction For PEDF Gene Expression In RPE Cells

Background Choroidal neovascularization is congenerous pathologic pattern of many fundus ocular disease. Almost develop to blind irreversibly, it is a sign of poor sight or blind. To inhibit the angiogenesis in healthy ocular tissue efficiently and in time appear to play a key role in prevent loss of visual function.Many factors are contributor to the formation of Choroidal neovascularization. among them, unbalanced levels of angiogenic stimulators and inhibitors of angiogenesis have been suggested to play a major role. It is a every important aim to rebuild the balance. A great of studys have been done to investigate the methods of inhibition of neovascularization. Employ the viral vectors delivery PEDF gene into RPE cells and increase gene expression has obtained some achievement. But the safety of use of viral vector have caused worry among many scientists. As a new type gene vector, Microbubble-enhanced ultrasound for gene delivery will become a method with safe and effective.Objectives 1.To approach feasibility of Microbubble-enhanced ultrasound enhance PEDF gene expression; 2.To develop the method and technique of gene transfection mediated by Microbubble-enhanced ultrasound.Methods 1.To obtain fresh eye from the donator, cells Isolate by enzyme digestion and the morph be observed under inverted microscope; 2.Amplification the hPEDF plasmid by E.coli DH5αstrain, identify and measure the concentration ; 3.optimization of screening. Detect cell survival rate and express rate after transfection of EGFP plasmid;4. Levels of PEDF protein are assayed by Western blot after 48hs hPEDF plasmid been transfected into RPE cells with optimization.Results 1.primary culture and serial subcultivation of RPE cells been established. cytokeratin immunochemistry proved the RPE cells; 2.the rate of transtection reach 10.6%,higher than the control group after ultrasound with 0.5W/cm2 intensity,work time 20s,microbubble concentration10% been applied on the RPE cells which the EGFP plasmid transfect into; 3. Our results show that ultrasound-mediated microbubble destruction on RPE cells is suitable for gene delivery and can enhance gene expression.Conclusions ultrasound with 0.5W/cm2 intensity,microbubble concentration10%,work time 20s are suitable for gene delivery and can enhance gene expression. The study provid a feasible data for animal experiment.