p53基因启动子转录活性检测细胞模型的建立

Establishment of Cell Model for Determining Transcriptive Activity of p53 Gene Promoter

【目的】构建p53基因启动子靶控报告基因细胞模型,为高通量筛选出以p53为靶标的中药提供新的细胞模型。【方法】采用瞬时转染报告基因测定法,确定最佳转染方案及p53基因启动子转录活性细胞模型的建立和验证,观察p53抑制剂PFT-a、血清饥饿、过氧化氢、自噬激活剂雷帕霉素对p53基因启动子转录活性PC12细胞模型的量效和时效关系。【结果】PFT-α诱导3 h,对p53基因启动子转录活性抑制率达50%左右;血清饥饿诱导12、24、36 h,p53基因启动子转录活性是对照组的1.6倍;过氧化氢300μmol/L诱导9 h,p53基因启动子转录活性是对照组的1.3倍;雷帕霉素在浓度为0.1、1 nmol/L可上调p53基因启动子转录活性,呈浓度依赖性,而5 nmol/L及更高浓度可下调p53基因启动子转录活性。【结论】成功建立采用p53基因启动子报告基因检测p53基因启动子转录活性的特异性细胞模型,血清饥饿、过氧化氢、雷帕霉素可诱导p53基因启动子的转录活性。

Objective PC12 cell model with p53 gene promoter target-controlled reporter gene was constructed for the establishment of cell model to screen p53-targeted Chinese herbal medicine. Methods By transient transfection of p53 promoter reporter gene, we optimized the transfection procedure, and established the PC12 cell model for determining the transcriptive activity of p53 gene promoter. After establishing the cell model, we investigated the dose-effect and time-effect relationship of transcriptive activity of p53 with p53 inhibitor PFT-tx, serum starvation, hydrogen peroxide, and autophagic activator rapamycin. Results Compared with the control group, the inhibition ratio of transcriptional activity of p53 gene promoter was about 50% after induction with PFT-oL for 3 hours. After serum starvation for 12, 24 and 36 hours, the transcriptional activity was 1.6 times as much as that of the control group, but the transcriptional activity was unchanged after serum induction for 24 and 36 hours. After induction with hydrogen peroxide at 300 ~mol/L, the transcriptional activity was 1.3 times as much as that of the control group. Rapamycin at the concentrations of 0. 1 and 1 nmoL/L up-regulated the transcriptional activity of p53 gene in concentration-dependent manner, while rapamycin at the concentration of 5 nmol/L and above down-regulated the activity. Conclusion The cell model for determining specific transcriptional activity of p53 with p53 promoter reporter genc has been established successfully, and serum starvation, hydrogen peroxide, and rapamycin can induce the transcriptive activity of p53 gene promoter.