p53蛋白在苯并（a）芘诱导的p21、E2F-1表达及细胞周期改变中的作用

Effects of p53 in benzo （ a ） pyrene induced p21 and E2F-1 expression and cell cycle changes

目的探讨p63蛋白在苯并（a）芘[benzo（a）pyrene，B（a）P]诱导的人胚肺成纤维细胞（HELF）细胞周期改变中的作用及其与p21、E2F-1的关系。方法转染p53siRNA质粒（p53-H）和载体CMV的HELF细胞（HELF／CMV）无血清培养48h后，加入2μmol／LB（a）P作用24h。用流式细胞仪检测细胞周期的变化。用Westernblotting检测细胞中p53、p21蛋白含量的改变，利用细胞核、细胞浆分离技术观察其亚细胞定位。用免疫荧光法观察E2F-1蛋白含量及细胞核、细胞浆分布。利用p53siRNA质粒和化学抑制剂pifithrin—α（PFT）抑制其表达，观察r63与p21、E2F-1的上下游关系以及其在B（a）P引起的细胞周期改变中的作用。结果2μmol／LB（a）P作用24h后，G1期细胞比例由（71±5）％减少为（39±4）％；p53、p21以及E2F-1蛋白含量增加，并且主要分布在细胞核内。用r63siRNA质粒和PFT抑制p53表达后，B（a）P诱导的p21高表达被抑制，细胞核内的含量明显减少；B（a）P诱导的e2F-1蛋白含量增加以及细胞周期的改变没有明显变化。结论B（a）P通过p53非依赖的信号通路引起HELF细胞周期的改变；p53对p21的表达具有调节作用，而对E2F-1的表达不具有调节作用。

Objective To investigate the roles of p53 in cell cycle changes on human embryo lung fihrohlasts （HELF） induced by benzo（a） pyrene[ B（a） P], and relationships between p53 and p21, E2F-1. Methods Cells transfected with p53 siRNA plasmid （p53-H） and CMV vector （HELF/CMV） were cultured with serum-free R/MINI-1640 for 48 hours, then treated with 2μmol/L B（a）P for 24 hours. Flow cytometry assay was used for detecting the cell cycle alteration after being exposed to B（a） P. Changes of p53 and p21 expressions were checked using Western blot assay, and the cytoplasmic and nuclear extraction was used to observe the subcellular localizations of p53 and p21. The immunofluorescence assay was used to check changes of E2F-1 expression and the distribution of E2F-1 in nuclear and cytoplasm after exposed to B （a）E p53siRNA plasmid and the chemical inhibitor of p53 [ pifithrin-ct （PFT） ] were used to observe effects of p53 in B（a） P induced cell cycle changes and the relationships of p53 and p21, E2F-1. Results After 2 μmol/L B（a）P exposure, the ratio ofGt phase cells （71±5）% was decreased to （39 ±4）% （P〈 0. 05）. p53, p21 and E2F-1 expressions were increased significantly, and over expressed proteins were mostly located in nuclear after B（a） P treatment. When p53 expression was inhibited by p53 siRNA or PFT, the decreases of G1 phase in response to B（a） P treatment still existed, and over expression of p21 induced by B（a）P was attenuated, especially in nuclear, hut E2F-1 over expression was not changed significantly. Conclusion B（a） P could induce cell cycle changes through p53 independent pathways. And p53 could regulate p21 expression positively, but not E2F-1.