抗氧化药物筛选细胞模型的建立.docVIP

 抗氧化药物筛选细胞模型的建立# 5 10 15 20 25 30 35 40 45  摘要：利用抗氧化作用元件(ARE）调控荧光素酶报告基因(Luc）的表达水平，从而建立抗 氧化药物筛选细胞模型。首先，构建由 ARE 调控荧光素酶报告基因表达的重组质粒载体 pARE-Luc-Neo，使用 FuGENE? HD 转染试剂将重组质粒转染人胚肾上皮细胞(Hek293 细 胞），通过 G418 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， 检测细胞中荧光素酶的活性，筛选荧光素酶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。观察不同浓度（0，3.125，6.25，12.5，25，50，100 μ mol/mL） 的 5 种中药单体穿心莲内酯、隐丹参酮、水飞蓟素、苦参碱和虎杖苷对荧光素酶报告基因表 达水平的影响。结果表明，通过检测荧光素酶活性筛选获得 Hek293-ARE，该细胞中荧光素 酶报告基因的表达水平受 ARE 调控，白藜芦醇和姜黄素实验表明表明成功建立了抗氧化药 物筛选细胞模型。5 种中药单体中穿心莲内酯、隐丹参酮与水飞蓟素有较好的诱导效果，且 在一定范围内与诱导剂的浓度呈剂量效应关系，表明所构建的细胞模型可用于抗氧化药物的 初步筛选。 关键词：细胞模型；抗氧化作用元件(ARE)；报告基因；药物筛选 中图分类号：Q813 Establishment of Screening Cell model for Antioxidant drugs LIU Yang1, CHEN Jangqing1, SHU Tejun1, LV ZhengBing1, ZHANG Yaozhou2 (1. Institute of Biochemistry, College of Life Science, Zhejiang Sci-Tech University; 2. Tianjin International Joint Academy of Biotechnology and Medicine) Abstract: To establish a screening cell model for antioxidant drugs by using antioxidant response element(ARE) regulates the expression levels of the luciferase reporter gene(Luc) . Recombinant plasmid vector pARE-Luc-Neo was constructed, which luciferase reporter gene expression regulated by the ARE. The recombinant plasmid vector transfected into human embryonic kidney epithelial cells(Hek293 cells) using FuGENE? HD transfection reagent. Cells were screened by G418, and positive monoclonal cells were selected by using the dilution method. The monoclonal cell lines with high activity of luciferase in response to antioxidant proteins inducer TBHQ was obtained by the detection of luciferase activity. Positive clones were treated with resveratrol and curcumin to detect the effect of screening. The effects of andrographolide, cryptotanshinone, silymarin, matrine and and polydatin at different concentration (0, 3.125, 6.25, 12.5, 25, 50, 100 μmol/L) were observed by determining reporter gene luciferase activity. The results show that Hek293-ARE cells were obtained through detecting luciferase activity, t