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目的利用抗氧化反应元件(ARE)调控荧光素酶(Luc)报告基因的表达水平,建立抗氧化药物筛选细胞模型。方法构建由ARE调控Luc表达的重组质粒载体pARE-Luc-Neo,使用FuGENE HD转染剂将重组质粒转染人胚肾上皮Hek293细胞,通过G418进行筛选,使用稀释法挑选阳性单克隆细胞,经抗氧化蛋白诱导剂TBHQ诱导,检测细胞中Luc的活性,筛选Luc高活性的单克隆细胞株。利用白藜芦醇和姜黄素评价阳性克隆的筛选效果,观察5个中药单体穿心莲内酯、隐丹参酮、水飞蓟素、苦参碱和虎杖苷在不同浓度(0、3.125、6.25、12.5、25、50、100μmol/L)时对Luc表达水平的影响。结果通过检测Luc活性,筛选获得Hek293-ARE,该细胞中的Luc的表达水平受ARE调控,且在一定范围内与诱导剂的浓度呈相关性。穿心莲内酯、隐丹参酮与水飞蓟素有较好的诱导效果。结论建立的抗氧化药物细胞筛选模型可有效地、高通量地用于抗氧化药物的初步筛选。
Objective To establish an anti-oxidative drug screening cell model by using the antioxidant regulatory element (ARE) to regulate the luciferase (Luc) reporter gene expression level. METHODS: The recombinant plasmid pARE-Luc-Neo was constructed by ARE-mediated Luc expression. The recombinant plasmid was transfected into human embryonic kidney epithelial Hek293 cells with FuGENE HD transfection reagent and then screened by G418. Positive monoclonal cells were selected by dilution method. The induction of TBHQ, an antioxidant protein inducer, detected the activity of Luc in cells and screened the monoclonal cell lines with high activity of Luc. The resveratrol and curcumin were used to evaluate the screening effect of positive clones. The effects of 5 Chinese herbal medicines, such as andrographolide, cryptotanshinone, silymarin, matrine and cediid, were evaluated at different concentrations (0, 3.125, 6.25, 12.5, , 100μmol / L) on the Luc expression level. Results Hek293-ARE was screened by detecting the activity of Luc. The expression level of Luc in this cell was regulated by ARE and correlated with the concentration of inducer within a certain range. Andrographolide, cryptotanshinone and silymarin have a better induction effect. Conclusion The established anti-oxidative drug cell screening model can be used effectively and high-throughput for primary screening of anti-oxidant drugs.