Experimental Research Of RB94 Gene Transfected Into The Retinoblastoma Of Nude Mices By Ultrasound-Targeted Microbubble Destruction

Background and Objective Retinoblastoma (RB) is the most common malignant intraocular tumor that occurs in developing retina in children. Nowadays, the therapy methods include ophthalmectomy, orbital exenteration, cryotherapy, charged-particle ratiotherapy,episcleral plaque ratiotherapy,transpupillary thermal therapy, et al. The past 20 years there were significant progress in the treatment of RB,gene therapy is in 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 vivo study, we detected the transfection efficiency of RB94 in retinoblastoma of nude mices by ultrasound-targeted microbubble destruction and explored the effection of RB94 gene on retinoblastoma.PART ONE THE DIFFERENCE OF ESTABLISH AN ANIMAL MODEL OF RETINOBLASTOMA BY SUBRETINAL INJECTION AND INTRAVITREAL INJECTIONMethods Rb cell line, HXO-Rb₄₄, was cultured in modified RPMI-1640 containing 10% fetal bovine serum and the cell suspension was prepared with the cell density (5.0-6.0)×10⁶/mL. Cell suspension was subretinally or intravitreously injected in 15 BALB/C (nu/nu) nude mice respectively to establish the Rb models. The growth of tumor in the eyes of nude mice were observed once a day after the injection of HXO-Rb44 cells under the slim lamp. Tumor-formating rate in the mice of different injection fashion groups were compared at 21, 28, 35 and 42 days after injection upon the histopathological diagnosis of the tumor. The identification of Rb were performed by the label of neurone specific enolase(NSE), S protein(S100) and glial fibrillary acidic protein(GFAP).Results The Rb grew in eyes after subretinal injection and intravitreous injection of suspension and gradually enlarged with the prolong of time. The tumor-formating rate was significantly increased in subretinal injection group comapred with intravitreous injection group at 21, 28, 35 and 42 days after injection, showing the statistically significant differences (P<0.05). The Rb cells presented the positive response for NSE, S100 and GFAP, showing the brown-yellow staining in the cytoplasm with the strongest reaction in NSE and weakest one in GFAP.Conclusion The subretinal injection of Rb suspension has a higher tumor-formating rate than intravitreal injection. The Rb animal model by subretinal injection of Rb suspension is reliable, and the biological behavior of Rb mouse model is resemble to one of clinical patient.PART TWO THE DIFFERENCE OF RB94 GENE TRANSFECTION EFFICIENCY OF DIFFERENT GENE VECTORMethods 32 BALB/C (nu/nu) nude mice were transplanted with HXO-Rb44 cells by subretinal injection. All successful animals modles were randomly divided into 6 groups:â‘ blank control group,â‘¡microbubbles+ultrasound,â‘¢RB94 plasmid+microbubbles,â‘£RB94 plasmid+liposome,⑤pEGFP-C1 plasmid+ microbubbles+ultrasound,â‘¥RB94 plasmid+microbubbles+ultrasound. The animals were sacrificed in the 7 days after transfection, then removed the eyeballs, the expression of RB94 gene was detected by RT-PCR.Results The results indicated that agarose gel electrophoresis lane were brighter in RB94 plasmid+microbubbles+ultrasound group than the RB94 plasmid+liposome group, and the difference between the two groups was statistically significant (P<0.05).Conclusion As a new gene vector, ultrasound-microbubble can raise the transfection efficiency of RB94 compared with traditional methods.PART THREE THE GROWTH ACTIVITY OF RETINOBLASTOMA AFTER RB94 GENE TRANSFECTION BY DIFFERENT METHODSMethods 32 BALB/C (nu/nu) nude mice were transplanted with HXO-Rb44 cells by subretinal injection. All successful animals modles were randomly divided into 6 groups same to part two. The animals were sacrificed in the 7 days after transfection, then removed the eyeballs, protein expressions of VEGF, MICA and MICB were analyzed by Western blot.Results Western blot analysis showed that the VEGF expression of RB94 plasmid+microbubbles+ultrasound group and Rb94 plasmid+liposome group were significantly lower than the other four groups, and the difference between the two groups was statistically significant (P<0.05). There was strong expression of MICA in RB94 plasmid+microbubbles+ultrasound group and RB94 plasmid+liposome group, and the difference between the two groups was statistically significant (P<0.05). The trends of expression of MICB were similar to MICA of each group, but the expression of MICB were slightly weaker than MICA .Conclusion RB94 gene could inhibit the activity of VEGF and improve the expression of MICA and MICB so that the NK cells'killing activity was improved, which suggested that RB94 transgene expression with ultrasound-targeted microbubble destruction could inhibit the growth of RB, which provided a reference of gene therapy for clinic.