超声微泡介导Rb94联合野生型p53基因对鼠视网膜母细胞瘤的抑制  

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

作　　者：高睿骐[1] 周希瑗[1] 杨映雪[1] 王志刚[2] 

机构地区：[1]重庆医科大学附属第二医院眼科,400010 [2]重庆医科大学超声影像研究所,400016

出　　处：《中华实验眼科杂志》2012年第10期908-913,共6页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金项目（30872826）

摘　　要：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／e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0．5W／cm^2。超声波辐照眼球4S，间隔24S，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wtp53mRNA及Rb94mRNA的表达；采用Western blot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wtp53mRNA及Rb94mRNA的表达条带；wtp53组wtp53mRNA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Rb94mRNA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�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 eo-transfeetion of Rb9d and wtp53 gene into subretina on RB with ultrasound mierobubble. Methods HXO-Rb44 suspension was subretinally injected to establish the RB model in 40 SPF female Balb/e 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 microvesiele 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/cm2 ultrasonic wave irradiated the eyeballs immediately for 4 seconds x2 times and interrupted for 24 seconds. Eyeballs were extracted 7 days after gene transfection, and the expressions of wtp53 mlRNA and Rb94 mRNA in tumor tissue 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 value 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 = O. 000 ）. Rb94 mRNA relative value was O. 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 wtp53 group, Rb94 group and wtp53 ＋Rb94 group, lm

关 键 词：基因转染/Rb94 野生型P53 超声微泡造影剂 视网膜母细胞瘤 凋亡 

分 类 号：R739.7[医药卫生—肿瘤]