Experimental Research Of Wild-type P53 Plasmid Transfected Into Retinablastoma Cells By Ultrasound Microbubble Intensifier

Background and Objective Retinoblastoma is the commonest intraocular malignant tumor in children. Nowadays, the therapy methods include picking off eyeball, laser photocoagulation, transpupillary thermotherapy, cryotherapy, ratiotherapy et al. Gene therapy for RB is still at the starting stage. As a new gene vector, microbubble can break up and release the plasmid it carries at the appointed place by ultrasound. In the study, we detected the transfection efficiency of wtp53 in Y79 cells that was mediated by ultrasound-microbubble and explored the optimum parameter of microbubble-ultrasound.PART ONETHE INFLUENCE OF ULTRASOUND USED TO Y79 CELLSMethods Devided Y79 cells into 6 groups, three of which were given ultrasound from 0.5 to 1.0 W/cm2 and the irradiation time were all 60 s, the other groups were given ultrasound from 0.5 to 1.0 W/cm2 and the irradiation time were all 30 s. Stained with 0.4% trypanblau for 4 min, then counted the stained cells and unstained cells.Results The survival rates of Y79 cells exposed to ultrasound with intensity of 0.5 and 0.75 W/cm2 (30 s) were beyond 90%, the ones exposed to ultrasound with the intensity of 0.5 and 0.75 W/cm2 (60 s) were all below 70%, and the rest ones exposed to ultrasound with the intensity of 1.0 W/cm2 (60 s or 30 s) were all below 50%.Conclusion When the ultrasound intensity were 0.5 and 0.75 W/cm2 (30 s), the survival rate of Y79 cells was not been affected, and was higher in 0.5 W/cm2 than 0.75 W/cm2 .PART TWOTHE DIFFERENCE OF WTP53 TRANSFECTION EFFICIENCY OF DIFFERENT GENE VECTORMethods Y79 cells were divided into three groups and each group was transfected with different methods. The plasmid-microbubbles-ultrasound was the first group, plasmid-liposome was the second group, and the plasmid-ultrasound took as the third group. After 24 to 48 hours, the wtp53 expression in the Y79 cells were detected by RT-PCR.Results The results indicated that agarose gel electrophoresis lane were brighter in the first group than the second group, while the third group had unsatisfactory transfection efficiency.Conclusion As a new gene vector, ultrasound-microbubble can raise the transfection efficiency of wtp53 compared with traditional methods.PART THREETHE GROWTH ACTIVITY OF Y79 CELLS AFTER WTP53 TRANSFECTED IN DIFFERENT METHODSMethods Y79 cells were divided into four groups and each one was transfected with different methods. The first three groups were the same as the second part, and the fourth group was blank control group. After 24 to 48 hours, the growth activity of Y79 cells were detected by flow cytometer.Results The results indicated that the growth activity of the first group was 25.58%, the second group was 19.50%, and the other two groups were lower than10%.Conclusion Wtp53 has obvious antiblastic effect towards Y79 cells, and the apoptosis rate of Y79 cells was the highest after transfection mediated by ultrasound-microbubble.