基于报告基因的PPAR-α激动剂筛选体系的建立

Establishment of a PPAR-α Agonist Screening System Base on Report Gene

关键词:  PPAR-α  报告基因  转染  药物筛选

Key words:  PPAR-α  report gene  transfection  drug screening

摘要:

目的 基于PPAR-α受体的信号传递通路和利用双萤光素酶报告基因分析方法，建立稳定的PPAR-α激动剂体外筛选体系。方法 pcDNA3.1-PPAR-α、pGL3-PPRE-luc及pRL-CMV共转染293T细胞后，设0，0.1，1，10，50 μmol·L^-1非诺贝特不同浓度点进行干预后，测定每浓度点重复5个样本。对转染优化试验数据，比较均值大小选取最佳结果；对不同转染组之间的差异性比较，采用t检验和方差分析。结果 浓度为0.1，1，10，50 μmol·L^-1非诺贝特干预组相对诱导率分别为：0.82，1.29，1.72，1.94，表现有统计学差异(P<0.05)。结论 成功建立基于PPAR-信号通路及双萤光素酶报告基因分析方法的PPAR-α激动剂筛药系统。

Abstract:

OBJECTIVE To establish a PPAR-α agonist screening system using a PPAR chimera system and double luciferase reporter gene assay method. METHODS pcDNA-hPPAR-α, pGL3-PPRE-luc and pRL-CMV (0.67 μg each) were Co-transfected into 293T cell seeded in 96 wells plate, fenofibrate was added into different groups after 6 hours transfection, respectively, up to terminal concentration of 0, 0.1, 1, 10, 50 μmol·L^-1 each. Two luciferase active were determined using Dual-Glo lucifarase assay system after 24 hours incubation, and relative luciferase intensive was used to express induction activity. RESULTS There is significant deviation between the 0.1, 1, 10, 50 μmol·L^-1 concentration point of fenobibrate, the relative induction activity of them were 0.82, 1.29, 1.72, 1.94. CONCLUSION A PPAR-α agonist screening system base on PPAR chimera system and double luciferase reporter gene assay method is established successfully.