PIN1反义基因转染抑制人骨肉瘤细胞的增殖  

Transfection of PIN1 antisense oligonucleotides inhibited proliferation of human osteosarcoma cells

作　　者：熊文化[1] 陈安民[1] 郭风劲[1] 张衣北[1] 黄涛[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院骨科,武汉430030

出　　处：《肿瘤》2006年第1期32-36,共5页Tumor

摘　　要：目的观察以pIRES2-EGFP质粒为载体的PIN1反义基因转染对人骨肉瘤细胞增殖的抑制作用。方法不同量(0、20、50、100、200、250μL)的PIN1反义基因以pIRES2-EGFP质粒为载体包装后转染人骨肉瘤MG-63细胞,收集转染前后的培养细胞和上清液,MTT法观测转染前后细胞生长曲线;流式细胞仪观测细胞生长周期变化和细胞凋亡情况;Western blot法检测PIN1蛋白的表达变化;逆转录聚合酶链式反应(RT-PCR)检测PIN1mRNA表达变化。结果MTT法和流式细胞仪检测显示反义PIN1基因转染后,MG-63细胞生长受到抑制,且其凋亡被促进;Western blot结果表明转染反义PIN1基因的量越多,其PIN1蛋白表达量越低,测得空白对照组及不同量转染组的光密度比值分别为0.854±0.136、0.866±0.138、0.732±0.154、0.611±0.121、0.547±0.109、0.398±0.113、0.320±0.151,差异有显著性(P<0.05);RT-PCR检测其光密度比值分别为0.983±0.125、0.988±0.127、0.915±0.157、0.786±0.125、0.608±0.124、0.433±0.130、0.410±0.158,差异有显著性(P<0.05)。结论反义PIN1基因可封闭PIN1mRNA,使MG-63细胞表达的PIN1蛋白减少,从而抑制人骨肉瘤MG-63细胞增殖。Objective：To evaluate the inhibitory effects of PIN1 antisense oligonucleotides on the poliferation of human osteosarma cells. Methods ： Different doses of antisense PIN1 oligonucleotides （0,20, 50,100,200,250 μL） in plasmid pIRES2-EGFP vector were transfected into osteosarcoma MG-63 cells. The cultured cells and supernatant before and after transfection were collected. The cell proliferation was determined by MTT method. The cell cycle and apoptosis were detected by FCM. The PIN1 protein expression was detected by Western blot and its mRNA expression was detected by reversed transcriptional polymerase chain reaction （RT-PCR）. Results： MTT and FCM assays indicated that the transfection with antisense PIN1 oligonucleotides could inhibit proliferation of MG-63 cells and lead to cell apoptosis. Western blot analysis revealed that antisense PIN1 oligonucleotides inhibited PIN1 protein expression in a dose-dependent manner （ P d0.05）. The optical density was 0. 854±0. 136, 0. 866±0. 138,0. 732±0. 154,0. 611±0. 121,0. 547±0. 109,0. 398±0. 113,0. 320±0. 151 after transfection of 0,20,50,100, 200,250μL of antisense PIN1 oligonucleotides. Conclusion： The expression of PIN1 mRNA in MG-63 cells could be inhibited by antisense PIN1 oligonucleotides which reduced the expression of PIN1 protein and suppressed the proliferation of human osteosarcoma MG-63 cells.

关 键 词：骨肉瘤 异构酶类 基因 PIN1 转染 寡核苷酸类 反义 MG-63细胞 

分 类 号：R738.7[医药卫生—肿瘤]