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目的观察全反式视黄酸(ATRA)逆转苯并(a)芘诱导的人胚肺成纤维细胞(HELF)细胞周期紊乱过程中细胞周期素D1(cyclinD1)、细胞周期素依赖性激酶K4(CDK4)、转录因子E2F1和E2F4的作用。方法用0.1μmol/LATRA预处理细胞后,再用2μmol/L苯并(a)芘作用细胞,用Westernblotting方法测定其蛋白表达水平;建立稳定转染了反义cyclinD1质粒和反义CDK4质粒的细胞系,应用反义技术证明上下游关系;用流式细胞技术测定细胞周期。结果2μmol/L苯并(a)芘作用于HELF24h后,cyclinD1、E2F1蛋白表达水平明显增高,CDK4和E2F4蛋白表达水平无明显变化;用0.1μmol/LATRA预处理24h后,cyclinD1、E2F1蛋白表达水平明显下降;与相同作用的对照组相比,苯并(a)芘刺激反义cyclinD1或反义CDK4细胞后E2F1表达增加的幅度明显降低;与相同作用的对照组相比ATRA预处理的反义cyclinD1或反义CDK4细胞中,苯并(a)芘诱导的E2F1过表达水平降低的幅度明显降低;流式细胞术测定结果显示,苯并(a)芘诱导细胞从G1期进入S期,ATRA通过聚积G1期细胞而阻滞苯并(a)芘诱导的细胞周期进程。结论ATRA通过cyclinD1/E2F1途径阻断苯并(a)芘诱导的HELF的细胞周期改变。
OBJECTIVE: To investigate the effects of all-trans retinoic acid (ATRA) on the reversal of benzo (a) pyrene-induced apoptosis in human embryonic lung fibroblasts (HELF) cells during the process of cell cycle arrest. CyclinD1, CDK4), transcription factors E2F1 and E2F4. Methods The cells were pretreated with 0.1 μmol / L ofATRA and then treated with 2 μmol / L of benzo (a) pyrene. Western blotting was used to determine the protein expression level. Cells stably transfected with antisense cyclinD1 and antisense CDK4 plasmids Department, the application of antisense technology to prove the relationship between upstream and downstream; flow cytometry cell cycle determination. Results After treated with 2 μmol / L benzo (a) pyrene for 24 h, the expressions of cyclin D1 and E2F1 protein were significantly increased while the expressions of CDK4 and E2F4 protein did not change significantly. After pretreatment with 0.1 μmol / L ATRA for 24 h, the expressions of cyclinD1 and E2F1 protein (P <0.05). Compared with the control group with the same effect, the increase of E2F1 expression was significantly decreased after the stimulation with benzo (a) pyrene in antisense cyclinD1 or antisense CDK4 cells. Compared with the control group with the same effect, antisense to ATRA pretreatment The decrease of E2F1 level induced by benzo (a) pyrene was significantly reduced in cyclinD1 or antisense CDK4 cells. The results of flow cytometry showed that benzo (a) pyrene induced G1 phase to S phase and ATRA Blocking Benzo (a) pyrene-induced cell cycle progression by accumulating G1 phase cells. Conclusion ATRA blocks the cell cycle of HELF induced by benzo (a) pyrene through cyclinD1 / E2F1 pathway.