构建抑制NF-κB信号通路的药物筛选模型  

Drug Screening Model Construction Based on Inhibiting NF-κB Signaling Pathway

作　　者：张立鹏[1] 李成海[1] 刘明耀[1] 罗剑[1] 

机构地区：[1]华东师范大学生命科学学院生命医学研究所,上海200241

出　　处：《激光生物学报》2010年第5期580-586,共7页Acta Laser Biology Sinica

基　　金：上海市科委研发公共服务平台项目(06DZ22923);国家自然科学基金项目(30800653)

摘　　要：NF-κB已被证明在肿瘤和炎症过程中起到至关重要的作用。因此,建立抑制NF-κB信号通路的药物筛选模型至关重要。利用荧光素酶报告基因技术与蛋白印迹技术分别探索TNFα处理浓度及时间对NF-κB报告基因表达和NF-κB抑制亚单位IκBα降解的影响,进而构建NF-κB信号通路抑制剂药物筛选模型。实验结果表明,0.01 nmol/L TNFα作用24 h即能刺激HEK293T细胞中NF-κB报告基因较高水平的表达,且其表达量与TNFα的剂量和处理时间呈正相关性;0.01 nmol/L TNFα作用5 min即能使Panc-28细胞中IκBα明显降解,20min～30 min几乎降解完全,之后IκBα含量又开始增加。NF-κB阳性抑制剂藤黄酸验证表明NF-κB萤光素酶报告基因检测筛选体系和NF-κB抑制亚单位降解筛选体系两种体系稳定可行。结果证明,两种模型可以用于NF-κB信号通路抑制剂药物的筛选。NF-κB is known as a major transcription factor that plays an essential role in the development of inflammation and cancer. Therefore, it＇ s a promising strategy to set up a drug screening model by inhibiting NF-κB signaling pathway. The effects of the concentrations and processing time of TNFα were explored on the NF-κB-luc reporter gene expression and the degradation of IκBα via the luciferase assay and western blot assay. The results showed, the expression level of NF-κB-luc reporter gene, which directly related to the concentrations and processing time of TNFα, was highly enough by the stimulation of 0.01 nmol/L TNFα for 24 h in HEK293T cells. IκBα degraded notably at the density of 0.01 nmol/L after 5 min treatment with TNFα on the Panc-28 cancer cells, and almost completely degraded after 20 min - 30min. Both the NF-κB luciferase reporter gene screening model and NF-κB inhibitory subunit screening model are confirmed by the known NF-κB positive inhibitor gambogic acid. Taken together, these two models can be used to select related potential drugs which inhibit NF-κB signaling pathway.

关 键 词：NF—κB 药物筛选模型 TNFΑ IKBΑ 藤磺酸 

分 类 号：Q291[生物学—细胞生物学] R915[医药卫生—微生物与生化药学]