苯并(a)芘对不同时相人胚肺成纤维细胞阻滞作用

Cell cycle arrest of synchronized human embryonic lung fibroblast exposed to benzo(a)pyrene

目的研究低遗传损伤浓度苯并（a）芘（BaP）对人胚肺成纤维细胞（HELF）的细胞周期调控的影响。方法0.5%血清饥饿法及10%血清再刺激方法获得处于各细胞周期时相的HELF;分别以二甲基亚砜（DMSO）、2,10和50μmol/L BaP于血清再刺激后10-12,16-18和22-24 h作用HELF2 h;采用流式细胞术分析细胞周期分布;采用蛋白印迹法分析细胞周期调控蛋白Cyclin D、Cyclin E、Cyclin A、Cyclin B、P53、P21和P16表达。结果BaP针对血清再刺激后10-12,16-18和22-24 h作用均引起明显的S期细胞比例的下降;血清再刺激后10-12 h作用引起G0/G1期细胞比例增加,BaP低、中、高浓度组分别为36.79%,42.73%,43.28%,伴随Cyclin D表达明显下降;血清再刺激后16-18 h作用引起G2/M期细胞比例增加,BaP低、中、高浓度组分别为18.46%,23.55%,28.44%,伴随P53和P21表达增强;血清再刺激后22-24 h作用引起G0/G1期细胞比例增加,BaP低、中、高浓度组分别为38.92%,54.08%,51.69%，伴随P53和P21表达增强。结论BaP针对不同时相的HELF作用顺序激活了G1和G22种周期关卡监控机制，并产生相应的周期阻滞效应。

Objective To explore the effect of benzo（a） pyrene（Bap） on cell cycle of synchronized human embryonic lung fibroblast（HELF）.Methods Treatments of 0.5% serum starvation and 10% serum re-stimulation were used to obtain cells synchronisation in G0/G1,S and G2/M stage.HELF cells synchronized in specific phase after serum re-stimulation were exposed to dimethylsulfoxide,2,10 and 50 μmol/L BaP for 2 hours.Cell cycle distribution and cell cycle regulation proteins such as cyclin D,cyclin E,cyclin A,cyclinB,P53,P21 and P16 were detected 12hr after BaP exposure with FCM and Western Blotting methods,respectively.Results Serum starvation and re-stimulation induced cells arrest in different phase and cell cycle phase change obviously 24 hr after serum re-stimulation.Cyclins expression in HELF cells deceased from G0 phase in a time-dependent manner.BaP induced a decrease of HELF cells in S stage.HELF treated with Bap were arrested in G1 stage and the expression of cyclin D decreased distinctly after serum re-stimulation of 10-12 hr.HELF with Bap treatment 16-18 hr after serum re-stimulation exhibited inceases of G2 stage and P53,P21 expressions.The ratio of G0/G1 and the expressions of P53 and P21 increased in HELF treated with Bap 22-24 hr after the serum re-stimulation.Conclusion BaP activates both G1 and G2 checkpoints of HELF synchronized in specific phase and induces cell cycle arrest.