PDCD4基因在胶质瘤细胞系的稳定表达及其对肿瘤细胞生长的影响

Stable expression of programmed cell death 4 gene in glioma cell line and its influence on proliferation of tumor cells

目的:建立稳定表达程序性死亡4基因(programmed cell death4,PDCD4)的人神经胶质瘤U251细胞系,观察PDCD4基因对人神经胶质瘤细胞增殖及细胞周期的影响。方法:将构建好的携带PDCD4重组真核表达载体pEGFP-PDCD4转染U251细胞,经过G418筛选获得稳定细胞系;用RT-PCR及Western blotting检测PDCD4mRNA和蛋白的表达情况,通过锥虫蓝染色活细胞计数法及克隆形成实验检测外源PDCD4转染对细胞增殖和克隆形成能力的影响,以流式细胞术检测细胞周期。结果:成功建立稳定表达PDCD4的胶质瘤细胞U251-PDCD4。未转染的U251及空载体转染的U251细胞均不表达PDCD4,而pEGFP-PDCD4转染的U251-PDCD4细胞表达高水平的PDCD4mRNA和蛋白质;转染PDCD4基因的细胞生长速度明显减慢(P<0.01)、克隆形成率明显降低(P<0.01);细胞周期检测显示,转染PDCD4的细胞较其他两对照组细胞S期升高、G2/M期明显降低(P<0.05)。结论:PDCD4通过干扰细胞周期明显抑制胶质瘤U251细胞的细胞增殖及克隆形成能力。

Objective ： To establish a glioma cell line U251 stably expressing programmed cell death 4 （PDCD4） gene, and to observe the influence of exogenous PDCD4 gene on the proliferation and cell cycle of U251 cells. Methods： Recom- binant eukaryotic expression vector pEGFP-PDCD4 was transfected into human glioma cell line U251 by Lipofectamine 2000, and the U251 cells stably expressing PDCD4 were established by G418 selection. Reverse transcription polymerase chain reaction （ RT-PCR ） and Western blotting were employed to detect the expression of PDCD4 mRNA and protein. Furthermore, cell proliferation and colony forming ability were determined by cell counting and colony formation assay; the cell cycle was detected by FACS. Results： High expression of PDCD4 mRNA and protein was observed in U251 cells transfected with pEGFP-PDCD4, whereas no PDCD4 mRNA and protein expression was detected in the non-transfected and vector-transfected cells. Further, cells transfeeted with pEGFP-PDCIM grew more slowly and had lower colony formation rate than cells of the other two control groups （P 〈 0. 01 ）. Moreover, transfection of PDCD4 significantly reduced the number of cells at the GJM phase and increased the cells at the S phase （ P 〈 0.05 ）. Conclusion ： Tumor suppressor gene PDCD4 can effectively inhibit cell proliferation and colony formation by altering their cell cycle.