苯并芘诱导人胚肺成纤维细胞cyclinD1、CDK4和E2F-1/4的表达改变

Benzo(a)pyrene induced changes of cyclin D1,CDK4 and E2F-1/4 expression in human embryo lung fibroblasts

目的建立苯并芘诱导的具有转化细胞部分特征的人胚肺成纤维细胞（T-HELF）模型,并观察T-HELF细胞中cyclin D1、CDK4和E2F-1/4蛋白表达的改变.方法以200、100、50、25、5、1μmol/L的苯并芘处理人胚肺成纤维细胞（HELF）24小时,用噻唑蓝（MTT）比色法测定苯并芘的细胞毒性.根据MTT结果确定以100和200μmol/L苯并芘给HELF细胞染毒3次,染毒结束后培养6周观察细胞的形态变化,培养12周后观察其形态变化及软琼脂中细胞克隆的生长.用流式细胞仪检测T-HELF细胞周期的变化,用Western blot检测T-HELF细胞中cyclin D1、CDK4和E2F-1/4蛋白表达的改变.结果MTT结果显示苯并芘浓度在25～200μmol/L之间时,细胞存活率为78%～80%.苯并芘200μmol/L组染毒4周后细胞死亡.100μmol/L组染毒结束后培养6周,观察到细胞变大,核增大或多核;12周后,在软琼脂中可以生长为细胞克隆,说明HELF细胞具有了转化细胞的部分特征.T-HELF细胞在无血清培养时,细胞周期没有停滞.用Western blot检测发现和正常HELF细胞相比T-HELF细胞的cyclin D1表达明显增加,CDK4、E2F-1和E2F-4的表达没有明显变化.结论建立了苯并芘诱导的具有转化细胞部分特征的人胚肺成纤维细胞模型,可用于苯并芘致癌的分子机制研究.无血清培养时,T-HELF细胞周期没有停滞,和正常HELF细胞相比T-HELF细胞cyclin D1表达明显增加,cyclin D1可能与T-HELF细胞的快速增殖有关.

Objective To establish Benzo （a）pyrene-treated human embryo lung fibroblasts （HELF）, which were provided with some characteristics of transformed cells （T-HELF） ,and observe the changes of cyclin D1, CDK4 and E2F- 1/4 expression in T-HELF cells. Methods Using 200,100,50,25,5 and 1μmol/L B（a）P treated HELF cells for 24h. Using MTT detected the cytotoxicity of B（a）P. 100,200μmol/L B（a）P was chosen for the treatment of HELF cells. H ELF cells was treated with B（a）P three times. The identification of T-HELF was investigated. Changes of cell cycle and the expression of cyelin D1 ,CDK4 and E2F-1/4 were checked using the flow cytometer and Western blot analysis. Results The survival rate of HELF cells treated with 25 - 200μmol/L B（a）P was 78% - 80% . 4 w after B（a）P treatment,cells were all dead in 200μmol/L groups. 6w after 100μmol/L B（a）P treatment,the morphological changes could be observed. 12w after treatment,there were colonies of T-HELF cells in soft agar. In T-HELF cells, cyclin D1 expression was higher than normal HELF, and cell cycle progressed through G, to S without serum. There were not significant changes of CDK4 and E2F-1/4 expression in T-HELF cells compared with normal HELF. Conclusion The T-HELF was established. This model provided a potential tool for investigating the molecular mechanism of carcinogenesis of B （a）P. The higher expression of cyclin DI may contribute to the cell cycle changes of T-HELF cells.