摘要
目的建立基于报告基因和核转录因子-κB(nuclear factor-kappa B,NF-κB)信号通路的药物筛选模型,探讨不同化学结构类型的化合物对脂多糖(lipopolysaccharide,LPS)诱导的NF-κB信号通路的抑制作用。方法构建带有NF-κB靶序列和报告基因的诱导性表达载体(TATA-like promoter-NF-κB-secreted alkaline phosphatase,pTAL-NF-κB-SEAP),并将其转入人胚肾细胞(HEK293)中,考察模型的稳定性、时效、量效关系以及脂多糖对细胞的增殖作用。结果转染pTAL-NF-κB-SEAP的HEK293细胞中SEAP报告基因的表达受脂多糖的诱导并呈剂量与时间依赖关系,脂多糖对阳性克隆细胞无增殖作用,本模型的Z′因子为0.88。结论利用此模型通过测定分泌型碱性磷酸酶(secreted alkaline phosphatase,SEAP)报告基因的诱导表达水平可筛选NF-κB信号通路抑制剂。
Objective To establish a reporter gene-based screening model of nuclear factor kappa B（NF-κB） signal transduction and study the inhibitory effects of compounds on NF-κB stimulated by lipopolysaccharide （LPS） using this model. Methods A recombinant vector TATA-like promoter-NF-κB-secreted alkaline phosphatase （pTAL-NF-κB-SEAP） was constructed by inserting NF-κB responsive elements in front of promot- er of pTAL-SEAP vector, pTAL-NF-κB-SEAP was then transfected into human embryonic kidney （HEK293） cells. The stability of the model, the time-effect relationship, the dose-response relationship and the proliferating effect of LPS were observed. Results The expression levels of reporter gene SEAP was induced by LPS in a dose-response and time-effect relationship manners. The Z＇ factor value was 0.88. LPS had no proliferating effects on HEK293 cells. Conclusions This model may be used to screen antagonists of NF-κB signal transduction by measurement of luminescent value of SEAP.
出处
《沈阳药科大学学报》
CAS
CSCD
北大核心
2007年第10期639-643,共5页
Journal of Shenyang Pharmaceutical University
关键词
核转录因子
阿尔茨海默病
碱性磷酸酶
报告基因
脂多糖
NF-κB
Alzheimer＇s disease
secreted alkaline phosphatase
reporter gene
lipopolysaccharide
