背景 研究表明,野生型p53(wtp53)和Rb94基因对人视网膜母细胞瘤(RB)的生长均有抑制作用,这2个基因是诱导和维持细胞衰老信号通路中重要的参与基因,因此2个基因联合应用是否对RB的生长抑制效果更好是近来关注的问题. 目的 观察超声微泡介导Rb94联合wtp53基因转染裸鼠RB后对其凋亡的影响.方法 将HXO-Rb44细胞悬液种植至40只雌性SPF级BALB/c裸鼠视网膜下腔建立RB动物模型,造模成功的裸鼠按随机数字表法平均分为模型对照组、wtp53质粒组(含wtp53质粒的微泡悬液)、Rb94质粒组(含Rb94质粒)和wtp53 +Rb94质粒组(联合组)(含wtp53质粒及Rb94质粒),其中模型对照组不做任何处理,其他3个组造模后第7天均由鼠尾静脉注入含相应基因的微泡悬液后,每天以0.5 W/cm2超声波辐照眼球4s,间隔24 s,循环2次.转染基因超声辐照7d摘除肿瘤组织,利用半定量逆转录PCR(RT-PCR)法检测肿瘤组织中wtp53 mRNA及Rb94 mRNA的表达；采用Western Not法检测基因转染的肿瘤组织中wtp53及Rb94蛋白的表达；用免疫组织化学法测定肿瘤组织中血管内皮生长因子(VEGF)的表达；采用TUNEL法检测基因转染后肿瘤组织的凋亡情况,计算凋亡指数(AI). 结果 HXO-Rb44细胞悬液视网膜下腔注射后移植瘤构建的成功率为80％(32/40),常规组织病理学检查提示检测样本的细胞异型性明显.基因转染后7d,模型对照组无wtp53 mRNA及Rb94 mRNA的表达条带；wtp53组wtp53 mRNA相对值为0.65±0.07,wtp53+Rb94组为0.32±0.02,差异有统计学意义(t=11.743,P=0.000)；Rb94组Rb94 mRNA相对值为0.42±0.03,wtp53 +Rb94组为0.23 ±0.03,差异有统计学意义(t=5.041,P=0.001).wtp53、Rb94或wtp53+ Rb94转染后,各组可见与转染相应的蛋白表达条带,wtp53+ Rb94组可同时检测到wtp53及Rb94蛋白的表达条带,但模型对照组无任何基因反应条带.免疫组织化学染色表明,wtp53+Rb94组肿瘤细胞中VEGF阳性反应强度明显弱于wtp53组、Rb94组和模型对照组.wtp53 +Rb94组AI为37.35±2.14,明显高于模型对照组的0.46±0.05、wtp53组的5.05±0.80和Rb94组的6.43±1.02,差异均有统计学意义(t=-34.395、-28.206、-26.006,P＜0.01).结论 超声微泡造影剂可介导双基因联合转染RB移植瘤,且Rb94联合wtp53基因对RB细胞的促凋亡作用较单基因转染增强.%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.
展开▼
