CAJ | 학술논문

目的探讨在石英刺激的人胚肺成纤维细胞(human embryo lung fibroblasts,HELF)中细胞外信号调节蛋白激酶(ERK)、应激活化蛋白激酶(JNK),细胞周期蛋白DI(cyclin D1)通路在石英诱导的细胞周期改变中的作用.方法建立稳定转染转录因子AP-1荧光素酶报告基冈质粒的HELF系(HELF-AP-1)及AP-1荧光素酶报告基因质粒与丝裂素活化蛋白激酶(MAPK)显性失活突变体质粒(DN-ERK、DN-JNK和DN-p38)分别共转染的HELF系(简称DN-ERK、DN-JNK和DN-p38).将HELF-AP-1、DN-ERK、DN-JNK和DN-p38细胞分为对照组和石英组(共8组),各对照组不加任何处理,石英组用200 μg/ml石英处理HELF细胞24 h.用免疫印迹(Western blot)法和免疫荧光法检测cyclin D1、细胞周期蛋白依赖激酶4(CDK4)和E2F-4蛋白表达;采用显性失活突变体技术验证MAPKs信号转导通路的上下游关系及其与细胞周期的关系;用流式细胞术检测细胞周期变化.结果 HELF-AP-1+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-AP-1对照组的差异有统计学意义(P＜0.05).抑制ERK或JNK表达后,与HELF-AP-1对照组比较,DN-ERK+石英组和DN-JNK+石英组G1期细胞百分比无明显变化.抑制p38后,DN-p38+石英组G1期细胞百分率明显下降,与HELF-AP-1对照组的差异有统计学意义(P＜0.05).与HELF-AP-1石英组比较,DN-ERK+石英组和DN-JNK+石英组CDK4表达降低和E2F-4表达增多,而DN-p38+石英组CDK4表达和E2F-4表达没有改变.与HELF-AP-1+石英组比较,DN-ERK+石英组和DN-JNK+石英组cyclin D1表达降低,而DN-p38+石英组cyclin D1没有改变.结论 ERK和JNK通过cyclin D1和CDK4介导石英诱导的HELF的细胞周期改变,而石英诱导的细胞周期改变与p38无关.
목적 탐토재석영자격적인배폐성섬유세포(human embryo lung fibroblasts,HELF)중세포외신호조절단백격매(ERK)、응격활화단백격매(JNK),세포주기단백DI(cyclin D1)통로재석영유도적세포주기개변중적작용.방법 건립은정전염전록인자AP-1형광소매보고기강질립적HELF계(HELF-AP-1)급AP-1형광소매보고기인질립여사렬소활화단백격매(MAPK)현성실활돌변체질립(DN-ERK、DN-JNK화DN-p38)분별공전염적HELF계(간칭DN-ERK、DN-JNK화DN-p38).장HELF-AP-1、DN-ERK、DN-JNK화DN-p38세포분위대조조화석영조(공8조),각대조조불가임하처리,석영조용200 μg/ml석영처리HELF세포24 h.용면역인적(Western blot)법화면역형광법검측cyclin D1、세포주기단백의뢰격매4(CDK4)화E2F-4단백표체;채용현성실활돌변체기술험증MAPKs신호전도통로적상하유관계급기여세포주기적관계;용류식세포술검측세포주기변화.결과 HELF-AP-1+석영조G1기세포소점비례하강,S기세포소점비례승고,여HELF-AP-1대조조적차이유통계학의의(P＜0.05).억제ERK혹JNK표체후,여HELF-AP-1대조조비교,DN-ERK+석영조화DN-JNK+석영조G1기세포백분비무명현변화.억제p38후,DN-p38+석영조G1기세포백분솔명현하강,여HELF-AP-1대조조적차이유통계학의의(P＜0.05).여HELF-AP-1석영조비교,DN-ERK+석영조화DN-JNK+석영조CDK4표체강저화E2F-4표체증다,이DN-p38+석영조CDK4표체화E2F-4표체몰유개변.여HELF-AP-1+석영조비교,DN-ERK+석영조화DN-JNK+석영조cyclin D1표체강저,이DN-p38+석영조cyclin D1몰유개변.결론 ERK화JNK통과cyclin D1화CDK4개도석영유도적HELF적세포주기개변,이석영유도적세포주기개변여p38무관.
Objective To investigate the roles of mitogen-activated protein kinases (MAPK) on silica-induced cell cycle changes. Methods After cells were treated with 200 μg/ml silica, Western blot and Immunofluorescence assays were utilized to detect the expression of cyclin D1, CDK4 and E2F-4, Flow cytometry was used to detect cell cycle progression, the dominant negative mutants techniques were used to investigate silica-induced signal pathway and the effects of which in silica-induced cell cycle changes. Results After cells were exposed to 200 μg/ml silica 24 h, the results of present study showed the proportion of cells in G1 phases was decreased. Silica-induced cell cycle alternation was markedly impaired by stable expression of a dominant negative mutants of ERK or JNK, but not p38. It was found that ERK and JNK were involved in silica-induced cyclin D1 and CDK4 overexpression and the decreased expression of E2F-4. Conclusion ERK and JNK, but not p38, mediated silica-induced cell cycle changes in human embryo lung fibroblasts.