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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G2 2种周期关卡监控机制,并产生相应的周期阻滞效应。
Objective To investigate the effect of BaP on the cell cycle regulation of human embryonic lung fibroblasts (HELF). Methods HELF at different cell cycle phases was obtained by 0.5% serum starvation and 10% serum restimulation methods. The levels of HELF in DMSO, 2,10 and 50μmol / L BaP, , HELF2 h at 16-18 and 22-24 h. The cell cycle distribution was analyzed by flow cytometry. The expressions of cyclin D, Cyclin E, Cyclin A, Cyclin B, P53, P21 and P16 were analyzed by Western blotting . Results BaP induced a significant decrease in the proportion of S phase cells at 10-12, 16-18 and 22-24 h after re-stimulation of serum. The proportion of cells in G0 / G1 phase increased after 10-12 h of restimulation Low, medium and high concentration groups were 36.79%, 42.73% and 43.28%, respectively, accompanied by a significant decrease of Cyclin D. Expression of low, medium and high concentration of BaP increased after 16-18 h Group were 18.46%, 23.55% and 28.44%, respectively. The expression of P53 and P21 was enhanced. The proportion of cells in G0 / G1 phase increased after 22-24 h of restimulation, while the concentrations of BaP in low, medium and high concentration groups were 38.92% 54.08% and 51.69% respectively, with the expression of P53 and P21 enhanced. Conclusion BaP activates G1 and G2 periodic checkpoint monitoring mechanisms in response to different phases of HELF and produces the corresponding cycle arrest effect.