Inhibition of subretinal co-transfection of Rb94 and wild-type p53 gene on retinoblastoma by ultrasound microbubble in nude mouse

Abstract

Background Researches showed that wild-type p53(wtp53)and Rb94 genes inhibit the growth of retinoblastoma(RB),and these genes are involved in signal pathway in the induction and maintenance of cellular senescence.Thus the combination of two genes to inhibit growth of RB is concerned.Objective This study was to observe the inhibitory effect of the co-transfection of Rb94 and wtp53 gcnc into subretina on RB with ultrasound microbubble.Methods HXO-Rb44 suspension was subretinally injected to establish the RB model in 40 SPF female Balb/c nude mice.The RB models were randomized into model control group,wtp53 group,Rb94 group and wtp53+Rb94 group,and 0.1 ml relevant gene microvesicle suspension was injected via caudal vein in the different groups,but no any gene was used in the model control group.Seven days after modeling,followed by 0.5 W/cm2ultrasonic wave irradiated the eyeballs immediately for 4 seconds ×2 times and interrupted for 24 seconds.Eyeballs were extracted 7 days after gene transfection,and the expressions of wtp53 mRNA and Rb94 mRNA in tumor tissuc were detected by RT-PCR,and wtp53 and Rb94 protein in tumor tissue were assayed using Western blot.Immunochemistry was used to exam the VEGF expression,and TUNEL was used to evaluate the apoptosis of the tumor cells.Results The model successful rate after HXO-Rb44 suspension was 80％ (32/40)and obvious malformation cells were seen under the light microscope.In 7 days after gene transfection,no response band for wtp53 mRNA and Rb94 mRNA were found.The relative expression valuc of wtp53 mRNA was 0.65±0.07 in the wtp53 group,and that in wtp53+Rb94 group was 0.32±0.02,showing a significant difference between them (t =11.743,P =0.000).Rb94mRNA relative value was 0.42 ±0.03 in Rb94 group,and that in the wtp53 + Rb94 group was 0.23 ± 0.03,with a significant difference(t=5.041,P=0.001).The response bands of wtp53 and Rb94 proteins were seen in wtp53group,Rb94 group and wtp53+Rb94 group.Immunochemistry showed that the positive reactive intensity for VEGF in tumor tissue was obviously weaker in wtp53+Rb94 group than that in the wtp53 group,Rb94 group and model control group.Apoptotic index(Al) was 37.35±2.14 in the wtp53+Rb94 group,showing a significant increase in comparison with model control group (0.46 ± 0.05),wtp53 group (5.05 ± 0.80) and Rb94 group (6.43 ± 1.02) (t =-34.395,-28.206,-26.006,P＜0.01).Conclusions Ultrasound microvesicle enable double gene transfecting into RB tumor tissue,and Rb94 gene cooperation with wtp53 gene enhance the inhibitory effect on RB by promoting the apoptosis of RB cells. Key words: Gene transfection/Rb94, wild-type p53; Ultrasound microvexicle; Retinoblastoma; Apoptosis