基于报告基因的PPRE转录调节模型的建立及应用

Establishment of a reporter gene system for detecting peroxisome proliferator response elements transcription and its application

目的:建立基于报告基因的靶向PPRE的转录调节模型,并观察不同浓度的PPAR特异性配体对上述模型的影响。方法:PCR扩增获得乙酰辅酶A氧化酶(ACO)启动子上包含PPRE的片段,构建重组报告质粒pGL3-PPRE,将此质粒单独或与PPARa或γ的真核表达质粒(pSG5-PPARα、pSG5-PPARγ)共转染到HEK293细胞中,通过测定荧光素酶(Luc)活力来分析非诺贝特对PPARα激活的作用以及吡格列酮和15-脱氧前列腺素J2(15d-PGJ2)对PPARγ途径的影响。结果:在pGL3-PPRE和pSG5-PPARα共转染的HEK293细胞中,荧光素酶的表达受非诺贝特的诱导,并呈现一定的剂量依赖关系;在共转染pGL3-PPRE和pSG5一PPARγ的HEK293细胞中,荧光素酶的表达则受吡格列酮、15d-PGJ2的诱导,也呈现一定的剂量依赖关系。结论:在HEK293细胞中建立了基于报告基因的PPRE转录调节模型,用此模型可以进行PPARα或γ配体的筛选。

Objectives： To establish a cellular model for screening peroxisome proliferator response elements transcription based on reporter gene, and to test the effect of specific ligands of PPAR at different concentrations on the above model. Methods： PPRE-pGL3 vector was reconstructed and transfeeted into HEK293 cells with or without pSG5-PPARα or pSG5-PPARγ. The transfected cells were then treated with fenofibrate, pioglitazone and 15d-PGJ2, respectively. Results： The results showed that the expression levels of reporter gene in eo-transfeeted HEK293 cells were induced in a dose-dependent manner by the representative PPARs activators fenofibrate, pioglitazone and 15d-PGJ2, Condusion： A PPRE regulatory reporter system was established in HEK293 cells, which may be used to monitor and screen agonists of PPARs.