活化蛋白-1和细胞周期蛋白在石英诱导的细胞周期改变中的作用

目的 探讨在石英刺激的人胚肺成纤维细胞(HELF)中AP-1/细胞周期蛋白D1 (cyclin D1)通路在细胞周期改变中的作用.方法 建立稳定转染空载体PXJ的HELF系(HELF-PXJ)及空载体PXJ与反义cyclin D1质粒或反义细胞周期蛋白依赖激酶(CDK4)质粒分别共转染的HELF系(简称anti-D1和anti-K4),将HELF-PXJ、anti-D1和anti-K4细胞分为对照组和石英刺激组(共6组),各对照组不加任何处理,石英刺激组用200 μg/ml石英处理细胞24h.检测AP-1对石英诱导的cyclin D1、CDK和E2F-4表达改变的影响.AP-1的化学抑制剂姜黄素(curcumin) 20μmol/L预处理细胞1h后,200 μg/ml石英刺激24 h,将HELF分为4组,分别为HELF对照组、HELF+石英组、HELF+curcumin对照组、HELF+curcumin+石英组.用免疫印迹(Western blot)法和免疫荧光法检测cyclin D1、CDK4和E2F-1/4蛋白表达;运用反义RNA技术证明通路的上下游关系;用流式细胞术检测细胞周期变化.结果 200 μg/ml石英处理HELF细胞,cyclin D1和CDK4蛋白表达升高,E2F-4蛋白表达明显降低,而E2F-1蛋白的表达没有明显改变.HELF-PXJ+石英组G1期细胞所占比例下降,S期细胞所占比例升高,与HELF-PXJ对照组的差异有统计学意义(P＜0.05).抑制cyclin D1或CDK4表达后,与对照组比较,anti-D1+石英组和anti-K4+石英组G1期细胞和S期细胞百分比无明显变化.抑制AP-1活性,与HELF+石英组比较,HELF+curcumin+石英组cyclin D1和CDK表达均减低,E2F-4表达增多.结论200μg/ml石英可诱导cyclin D1和CDK4蛋白表达增高,E2F-4蛋白表达减少,并通过AP-1/cyclin D1通路诱导细胞周期改变.

The roles of activated protein-1 and cell cycle protein in silica-induced cell cycle changes

Objective To investigate the roles of cyclin D1 and CDK4 in the cell cycle changes of human embryonic lung fibroblasts (HELFs) exposed to silica.Methods HELFs were divided into 4 groups:control group,curcumin (20 pmol/L for 1 h) group,silica (200 μg/ml for 24 h) group and curcumin plus silica group,i.e.after exposure to 20 μmol/L curcumin for 1h,the HELFs were treated with 200 μg/ml silica for 24 h.Western blot and Immunofluorescence assays were utilized to detect the expression levels of cyclin D1,CDK4 and E2F1/4.Flow cytometry was used to detect the cell cycle progression,the RNA transfection technique was used to investigate the silica-induced signal pathway and the roles of which in silica-induced cell cycle changes.Results The expression levels of cyclin D1 and CDK4 significantly increased and the expression level of E2F-4 decreased obviously,but the expression level of E2F-1 did not significantly change in silica group.The proportion of G1 phase cells obviously decreased and the proportion of S phase cells significantly increased in silica group,as compared with control group (P＜0.05).When suppressing the expression of cyclin D1 or CDK4,the proportions of cells in G1 phase in anti-D1 plus silica group and anti-K4 plus silica group did not obviously change,as compared with control group.When suppressing AP-1 activity,the cyclin D1 and CDK4 expression levels decreased and the E2F-4 expression level increased in curcumin plus silica group,as compared with silica group.Conclusion The results of present suggested that 200 μg/ml silica could induce the high expression of cyclin D1 and CDK4 and the low expression of E2F-4,resulting in the cell cycle changes by AP-1/cyclin D1 pathway in HELFs.