沉默Robo基因对人膀胱移形上皮癌细胞T24增殖的影响  

Effect of Silent Robo Gene on Proliferation of Transitional Epithelial Carcinoma of Human Bladder

作　　者：王晗[1] 姚狮章 朱朝阳[3] 程晓晖[1] 田素民[1] 李国营[1] 

机构地区：[1]广东药科大学基础学院,广东广州510006 [2]佛山市南海区第四人民医院泌尿外科,广东佛山528211 [3]河南大学附属淮河医院泌尿外科,河南开封475000

出　　处：《中山大学学报（医学科学版）》2016年第4期514-521,共8页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：广东省"4百十"工程人才培养项目(2050205F-43-03)

摘　　要：【目的】研究Robo蛋白在人膀胱移形上皮癌细胞(T24)中的表达及沉默Robo基因对T24细胞增殖的影响。【方法】实验分为两组,对照组(不转染载体)和转染组(转染复合载体),每组20例。对照组体外培养T24细胞,用免疫组织化学、免疫荧光和蛋白质印迹检测是否有Robo蛋白的表达及其表达亚型;转染组利用RNA干扰技术,寻找表达基因的沉默位点,构建合理的表达载体,转染T24细胞,免疫荧光化学观察细胞转染结果,蛋白质印迹来检测该表达基因沉默后蛋白表达的变化,MTT检测该表达基因沉默后细胞存活率变化。【结果】1免疫组化和免疫荧光检测显示T24细胞中有Robo1和Robo4表达,且表达差异无统计学意义(P=0.363和P=0.241),而正常膀胱移行上皮细胞中无表达,蛋白质印迹证实了免疫组化结果(P=0.205)。2沉默Robo基因后,T24细胞中Robo1和Robo4的表达受到抑制,尤其是Robo1的表达几乎受到全部抑制(免疫荧光P=0.002,蛋白印迹P=0.0001),MTT法检测显示Robo1干扰组细胞的存活率明显下降,与对照组及Robo4组相比差异有显著性(分别为P=0.012和P=0.023)。【结论】T24细胞中存在Robo1和Robo4两种蛋白受体,沉默Robo基因后,Robo1的表达和T24细胞的存活率明显下降。[Objective] To study the expression of Robos in T24 ceils and the influence in human bladder transitional epithelial carcinoma T24 cell proliferation when Robo gene was silenced. [ Methods ] The experiment was divided into two groups：control group （without carrier） and interference group（with composite carrier）. （1）Immunohistochemistry, immunofluorescence and Western blot was performed to investigate whether Robos expressed in T24 cells cultured in vitro, and determined the types of this expression. （2） T24 cells were transferred by RNAi technique through seeking the gene silent sites and constructing a proper expression vector. Immunofluorescence was carried out to confirm the results of interference and Western blot was performed to detect the changes of expression of Robos. Survival rate was evaluated by MTT method. [ Results ] （1）Immunohistochemistry and immunofluorescence showed that the expression subtypes of T24 cells were Robol and Robo4 with no statistically significant （P = 0.363 and P = 0.241 ）, and none of the subtypes is expressed in normal transitional epithelial cancer cells. Western blot confirmed this result（P = 0.205）. （2） RNAi technique efficiently silenced the expressions of Robol and Robo4 in T24, especially Robol, almost was completely inhibited （immunofluorescence P = 0.002, Western blot P = 0.0001 ）. Survival rate of Robol-interfered group, had significant difference （P = 0.012 and P = 0.02） with control group and Robo4 group, decreased obviously as showed by MTT method. [Conclusion] Protein receptors Robol and Robo4 were demonstrated exist in T24 ceils and the expression of Robol and survival rate of T24 cells decreased obviously after Robo gene was silenced

关 键 词：人膀胱形上癌T24细胞 Robo蛋白 免疫荧光 免疫组化 蛋白质印迹 RNA干扰 

分 类 号：R737.14[医药卫生—肿瘤]