CAJ | 학술논문

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.
목적 탐토재석영자격적인배폐성섬유세포(HELF)중AP-1/세포주기단백D1 (cyclin D1)통로재세포주기개변중적작용.방법 건립은정전염공재체PXJ적HELF계(HELF-PXJ)급공재체PXJ여반의cyclin D1질립혹반의세포주기단백의뢰격매(CDK4)질립분별공전염적HELF계(간칭anti-D1화anti-K4),장HELF-PXJ、anti-D1화anti-K4세포분위대조조화석영자격조(공6조),각대조조불가임하처리,석영자격조용200 μg/ml석영처리세포24h.검측AP-1대석영유도적cyclin D1、CDK화E2F-4표체개변적영향.AP-1적화학억제제강황소(curcumin) 20μmol/L예처리세포1h후,200 μg/ml석영자격24 h,장HELF분위4조,분별위HELF대조조、HELF+석영조、HELF+curcumin대조조、HELF+curcumin+석영조.용면역인적(Western blot)법화면역형광법검측cyclin D1、CDK4화E2F-1/4단백표체；운용반의RNA기술증명통로적상하유관계；용류식세포술검측세포주기변화.결과 200 μg/ml석영처리HELF세포,cyclin D1화CDK4단백표체승고,E2F-4단백표체명현강저,이E2F-1단백적표체몰유명현개변.HELF-PXJ+석영조G1기세포소점비례하강,S기세포소점비례승고,여HELF-PXJ대조조적차이유통계학의의(P＜0.05).억제cyclin D1혹CDK4표체후,여대조조비교,anti-D1+석영조화anti-K4+석영조G1기세포화S기세포백분비무명현변화.억제AP-1활성,여HELF+석영조비교,HELF+curcumin+석영조cyclin D1화CDK표체균감저,E2F-4표체증다.결론200μg/ml석영가유도cyclin D1화CDK4단백표체증고,E2F-4단백표체감소,병통과AP-1/cyclin D1통로유도세포주기개변.
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.