苯并(a)芘对不同时相人胚肺成纤维细胞细胞周期的影响

Effects of Benzo(a)pyrene on Cell Cycle Distribution of the Human Embryonic Lung Fibroblast Synchronized at Different Phase

目的研究不同细胞周期状态下,苯并(a)芘[B(a)P]对人胚肺成纤维细胞周期的影响。方法采用血清饥饿的方法使人胚肺成纤维细胞(HELF)同步于G0期,血清再刺激后细胞较为同步地进入周期,分别于G1、S和G2-M期细胞占多数的情况下对细胞进行B(a)P(2、10、50μmol/L)染毒处理,染毒方式分B(a)P经代谢活化和未经代谢活化两种。结果血清饥饿48h,细胞较好地同步于G0期,血清再刺激后10～12h、16～18h、22～24h为G1、S和G2-M期改变明显的时间。未经代谢活化的B(a)P对细胞周期的影响较弱,改良的代谢活化方法既能较好地代谢活化B(a)P,又尽可能避免了对细胞周期的干扰作用。除2μmol/L组在22h引起G1期细胞百分比下降和S期细胞百分比增加外,其他时点和剂量均有S期百分比下降的效应,随着剂量增加,该效应越明显,16h改变最明显;16h出现G2-M期细胞百分比增加的效应;10和22h出现G1期细胞百分比增加和G2-M期细胞百分比下降的效应。结论血清饥饿及再刺激方法能得到处于各时相的细胞;改良的代谢活化方法适合细胞周期研究;B(a)P在实验周期均能引起HELFS期细胞减少,作用于G1期可引起G1期阻滞,作用于S期可引起G2-M阻滞,作用于G2期可引起G1阻滞。

Objective To evaluate influence of benzo（a）pyrene on cell cycle of the human embryonic lung fibmblast at different synchronous stage. Methods Cell cycle distribution were detected by flow cytometry（FCM）. Cells were synchronized at cycle GO stage through 48 hours serum starvation and reenter cell cycle together at a synchronous state after resupplied with whole growth medium contain good serum. Benzo（a）pyrene with a series concentrations were directly used or metabolized active by traditional and modified metabolize active methods through preincubation of $9 and were treated as regent to HELF cells mostly synchronized at G1, S or G2-M stage respectively. Then cell cycle distribution changes were detected 12 h after treatment. Results Serum starvation （48 hours） could synchronize HELF at G0-G1 stage effectively and 10-12 h, 16-18 h, 22-24 h were cycle phase change distinctly time after serum restimulated. Cells were synchronized at Gl, S and G2-M stage mostly. Benzo（a）pyrene influence cycle distribution little without metabolize active, while modified methods could metabolize active benzo（a）pyrene well and avoided disturbing effect of S9 on cell cycle in traditional method. Except 2 μmol/L benzo（a）pyrene treated at 22 h after restimulated caused the percentage of cells at S stage increase, most treatment induced the percentage of cells at S stage decrease which was associated obviously with the increasing dosages. The percentage of cell at S stage decrease at 16 h were more remarkable than other times and the percentage of cell at G2-M increase correspondingly. While the percentage of cell at Gl increase and the percentage of cell at G2-M decrease were obsered when benzo（a）pyrene treated at 10h and 22h after restimulated. Conclusion Serum starvation 48 hours and restimulate can synchronize HELF at different stage effectively. Modified metabolize active method is suitable for cell cycle research. Primary influence of benzo（a）pyrene on cell cycle is the decreased percentage of cells at S stage, Gl arrest, G2-M arrest and G1 arrest were occurred when benzo（a）pyrene were treated at Gl, S and G2 stage respectively.