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目的探讨JWA在化学致突变物烷化剂N-甲基-N’-硝基-N-亚硝基胍(MNNG)诱导人支气管上皮(HBE)细胞凋亡中的作用及其可能机制。方法用噻唑蓝(MTT)法检测细胞生长抑制率,用Hoechst染色法检测细胞凋亡,用Western blot法检测JWA蛋白表达,用Southwestern印迹分析法检测JWA基因近端启动子的结合蛋白。结果MNNG处理HBE细胞24 h均可诱导细胞发生凋亡,并呈现明显的剂量-效应关系；MNNG诱导HBE细胞凋亡过程中伴随着JWA蛋白表达升高。进一步用2．0μg/ml MNNG处理HBE细胞24h后,用Southwestern方法从HBE细胞核蛋白中检测出一与JWA近端启动子能特异性结合的转录因子。结论MNNG激活HBE细胞中核转录因子与JWA近端启动子结合后可能激活了细胞内凋亡的信号通路。
Objective To investigate the role of JWA in the apoptosis of human bronchial epithelial cells (HBE) induced by chemical mutagenic alkylating agent N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) and its possible mechanism. Methods Cell growth inhibition rate was detected by MTT assay. Apoptosis was detected by Hoechst staining. JWA protein expression was detected by Western blot. The binding protein of JWA gene proximal promoter was detected by Southwestern blotting. Results MNNG treatment of HBE cells induced apoptosis in 24 h, and showed a dose-response relationship. MNNG induced apoptosis in HBE cells accompanied by an increase in JWA protein expression. Further treatment of HBE cells with 2.0 μg / ml MNNG for 24 h detected a transcription factor specific for the JWA proximal promoter from the HBE nucleoprotein using the Southwestern method. Conclusion MNNG activation of HBE cells in nuclear transcription factor and JWA proximal promoter may activate intracellular apoptosis signaling pathway.