[关键词]
[摘要]
摘 要 目的: 建立稳定表达程序性死亡4基因(programmed cell death 4, 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PDCD4 mRNA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PDCD4 mRNA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。
[Key word]
[Abstract]
Abstract Objective: To establish a glioma cell line U251 stably expressing programmed cell death 4(PDCD4) gene, and to observe the influence of exogenous PDCD4gene on the proliferation and cell cycle of U251 cells.Methods: Recombinant eukaryotic expression vector pEGFPPDCD4 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 (RTPCR) and Western blotting were employed to detect the expression ofPDCD4 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 pEGFPPDCD4, whereas no PDCD4 mRNA and protein expression was detected in the nontransfected and vectortransfected cells. Further, cells transfected with pEGFPPDCD4 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 G2/M 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.
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[基金项目]
山东省中青年科学家科研奖励基金(No.2006BS03064); 中国博士后科学基金(No.2006BS03064).