一种基于抗氧化反应元件的抗氧化药物筛选模型的制备

Establishment of a drug screening model based on anti-oxidative response element

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 a cell model for anti-oxidative drug screening by using anti-oxidative response element （ARE） to regulate the expression level of luciferase （Luc） reporter gene. Methods The recombinant plasmid vector pARE-Luc-Neo, expressed by ARE-regulated Luc reporter gene, was constructed. The recombinant plasmid vector was transfected into human embryonic kidney epithelial cells （Hek293 cells） using FuGENE liD transfection reagent. The positive cells were screened by G418, and the positive monoclonal ceils were selected using the dilution method. The monoclonal cell lines with high activity of Luc reporter gene in response to anti-oxidative protein inducer TBHQ were obtained using the Luc activity detection. The screening effect of the positive clones was evaluated by resveratrol and curcumin. The effects of andrographolide, cryptotanshinone, silymarin, matrine, and polydatin at different concentration （0, 3.125, 6.25, 12.5, 25, 50, and 100 μmol/L） on Luc expression level were observed. Results The Hek293-ARE ceils were obtained by detecting the Luc reporter gene activity, the Luc reporter gene expression was regulated by ARE, meanwhile there exists a dose-response relationship between the expression level and the concentration of the inducer within a certain range. The andrographolide, cryptotanshinone, and silymarin have significant induction effects. Conclusion The cell model for anti-oxidative drug screening is established successfully and could be effectively and high-throughput for the preliminary screening of anti-oxidative drugs.