神经胶质瘤致病因子-1在肾癌细胞株769-P体外侵袭活性强化中的作用及其机制  

Effect and mechanism of glioma-associated oncogene homolog-1 on the activity of 769-P in renal cell carcinoma cell line in vitro

作　　者：杨庆良[1] 张海芳[1] 张广云[1] 周晨龙[1] 

机构地区：[1]河南省安阳市人民医院泌尿外科,455001

出　　处：《中华实验外科杂志》2016年第4期1037-1040,共4页Chinese Journal of Experimental Surgery

摘　　要：目的 观察神经胶质瘤致病因子-1（Gli-1）在肾癌细胞株769-P细胞体外侵袭活性强化中的作用.方法 构建Gli-1-小干扰RNA （siRNA）慢病毒载体,将体外培养好的肾癌769-P细胞株分为3组：A组：Gli-1-siRNA和pGCL-绿色荧光蛋白（GFP）共转染组.B组：未转染组;C组：pGCL和pHelper 1.0共转染组.A组为实验组,B、C两组为对照组.应用基因转染方法,将A组肾癌769-P细胞转染Gli-1-siRNA慢病毒载体.检测Gli-1基因表达对肾癌细胞株769-P细胞的抗增殖作用和生物学功能的影响,通过Western blot和实时定量聚合酶链反应（Real-time PCR）技术检测Gli-1-siRNA在769-P细胞中的表达水平,并通过噻唑蓝（MTT）法、划痕实验、流式细胞仪、显微镜等观察比较转染Gli-1-siRNA的769-P细胞的的迁移能力、黏附能力和凋亡等形态学改变.结果 （1）阴性对照组的Gli-1 mRNA表达量为98.2 ±6.6,Gli-1-siRNA转染组为53.8 ±4.6,差异有统计学意义（P＜0.05）.（2）阴性对照组的Gli-1蛋白表达量为94.9±7.9,Gli-1-siRNA转染组为42.6±3.2,差异有统计学意义（P＜0.05）.（3）划痕实验结果显示Gli-1-siRNA转染的769-P细胞株迁移能力低于正常769-P细胞株（P＜0.05）.（4） MTT实验结果显示,Gli-1-siRNA对769-P细胞的活性抑制明显增强,相同作用时间不同比例之间差异有统计学意义（P＜0.05）,同一比例不同作用时间之间差异有统计学意义（P＜0.01）.（5）流式细胞术检测结果显示,B、C组769-P细胞12 h的凋亡率为（10.67±0.23）％和（6.84±0.16）％,A组细胞12h凋亡率为（45.40±5.79）％,24h凋亡率为（60.67±5.79）％,与对照组比较差异有统计学意义（P＜0.05）.（6）A组Gli-1-siRNA转染成功的769-P细胞其胞质内出现颗粒状物,肾癌细胞逐渐模糊、消失.24h后769-P细胞数明显减少,48 h后癌细胞大部分凋亡,细胞漂浮,并且在培养液中出现细胞碎片.B、C组769-P细胞状况良好,未见明显细胞�Objective To investigate the effect of glioma-associated oncogene homolog^-1 （Gli-1） on the invasion of renal cell carcinoma cell line 769-P in vitro.Methods Gli-1-small interfering RNA （siRNA） lentiviral vector was constructed,and 769-P cells in vitro cultured were divided into three groups：group A [Gli-1-siRNA and pGGL-green fluorescent protein （GFP） co-transfection];group B （non transfection）,and group C （pGCL and phelper 1.0 co-transfection）.Using gene transfection method,the 769-P cells in group A were transfected with Gli-1-siRNA lentiviral vector vector.Gli-1-siRNA gene was constructed to detect the effect of Gli-1 gene on the proliferation and biological function of 769-P cells.The expression level of Gli-1-siRNA in 769-P cells was detected by real-time quantitative polymerase chain reaction （Real-time PCR） and Western blotting,and the migration ability of 769-P cells was observed by methyl thiazol tetrazolium （MTT） assay,flow cytometry and microscope.Results （1） In group B and A,the expression of Gli-1 mRNA was 98.2 ± 6.6 and 53.8 ± 4.6 with the difference being statistically significant （P 〈 0.05）.（2） In group B and group A,the expression of Gli-1 protein was 94.9 ± 7.9 and 42.6 ± 3.2 with the difference being statistically significant （P 〈 0.05）.（3） Scratch test showed that the migrative ability of 769-P cells transfected with Gli-1-siRNA was significantly reduced as compared with normal 769-P cells （P 〈 0.05）.（4） MTT assay showed that siRNA Gli-1 significantly inhibited the activity of 769-P cells in a time-and concentration-dependent manner （P 〈 0.05）.（5） Flow cytometry showed that the apoptosis rate of 769-P cells in groups B and C was （10.67±0.23）% and （6.84±0.16）%,and that in group A at 12 h and 24 h was （45.40 ± 5.79） % and （60.67 ± 5.79） % respectively with the difference being statistically significant （P 〈 0.05）.（6） The cytoplasm of 769-P cells in group A was granular,and 769-P cells gra

关 键 词：肾癌 神经胶质瘤致病因子-1 小干扰RNA 侵袭 

分 类 号：R737.11[医药卫生—肿瘤]