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目的研究苯并(a)芘[B(a)P]对人胚肺成纤维细胞(HELF)的细胞周期分布及细胞周期蛋白D1(cyclin D1)和细胞周期蛋白依赖激酶4(CDK4)蛋白表达的影响,并探讨两种蛋白含量改变与细胞周期效应之间的关系。方法将反义cyclin D1质粒和反义CDK4质粒导入HELF细胞内,建立两种质粒稳定转染的细胞模型。用0.1、0.5、2.5和12.5μmol/L的B(a)P处理HELF细胞24h,用蛋白印迹方法检测cyclin D1和CDK4蛋白表达水平;利用流式细胞技术检测B(a)P处理对HELF细胞及两种稳定转染细胞系细胞周期的影响。结果成功建立了反义cyclin D1和反义CDK4稳定转染的细胞系。不同剂量B(a)P处理可引起cyclin D1蛋白表达的显著增加,但对CDK4蛋白表达无明显影响;2.5μmol/L的B(a)P处理HELF细胞24h后,引起其细胞周期G1期显著下降,S期显著增加;2.5μmol/L的B(a)P处理反义cyclin D1和反义CDK4稳定转染的HELF细胞24h后,对其细胞周期的分布无显著影响。结论Cyclin D1和CDK4基因均参与了B(a)P所致细胞周期改变过程,并发挥正性调节作用。
Objective To investigate the cell cycle distribution and expression of cyclin D1 and CDK4 in human embryonic lung fibroblasts (HELF) induced by benzo (a) pyrene [B (a) P] , And to explore the relationship between changes in the two protein levels and cell cycle effects. Methods Antisense cyclin D1 and antisense CDK4 plasmids were introduced into HELF cells to establish two cell lines stably transfected with plasmids. HELF cells were treated with 0.1, 0.5, 2.5 and 12.5 μmol / L of P (a) P for 24 h, Western blotting was used to detect the expression of cyclin D1 and CDK4 protein. Flow cytometry was used to detect the effect of B (a) P treatment on HELF cells And the cell cycle of two stably transfected cell lines. Results Antisense cyclin D1 and antisense CDK4 stably transfected cell lines were successfully established. Treatment with different doses of B (a) P resulted in a significant increase of cyclin D1 protein expression but no significant effect on CDK4 protein expression. After treatment with 2.5 μmol / L B (a) P for 24 h, the cell cycle G1 significantly increased (P <0.05). After treatment with 2.5 μmol / L B (a) P for 24 h, the HELF cells stably transfected with antisense cyclin D1 and antisense CDK4 had no significant effect on the cell cycle distribution. Conclusion Both Cyclin D1 and CDK4 genes are involved in the cell cycle change induced by B (a) P, and play a positive regulatory role.