基于报告基因检测的PXR、FXR和LXRα激动剂高通量筛选模型的建立  

Establishment of cell models for PXR,FXR and LXRα agonists high-throughput screening based on reporter gene assay

作　　者：庄嘉琅[1] 曾行[1] 钟国平[1] 金晶[1] 苟晓丽[1] 毕惠嫦[1] 黄民[1] 

机构地区：[1]中山大学药学院药物代谢与药动学实验室,广东广州510006

出　　处：《中国药理学通报》2015年第2期289-293,共5页Chinese Pharmacological Bulletin

基　　金：广东省新药设计与评价重点实验室开放基金(No 2011A060901014-009)

摘　　要：目的建立基于报告基因法的高通量筛选细胞模型,用来发现PXR、FXR和LXRα受体激动剂。方法利用Real-time定量PCR方法比较HEK293、Hep G2和LS174T细胞中内源性核受体PXR、FXR和LXRα的表达量,将p SG5-h PXR和p GL3-XREM-CYP3A4、p EGFP-N3-h FXR和EcRETK-Luc、p CMX-FLAG-h LXRα和p GL3-XREM-CYP3A4等质粒分别共转染到工具细胞中,优化共转染比例,并考察阳性药与萤光素酶报告基因表达强度的量效关系、模型特异性和稳定性。结果 1根据Real-time定量PCR结果,模型选用低表达PXR、FXR和LXRα的HEK293细胞作为工具细胞;2根据不同共转染比例对报告基因活性的结果,PXR、FXR和LXRα报告基因药物筛选模型的报告基因和过表达质粒比例,最终分别选择1∶1、2∶1和2∶1;3模型中,报告基因活性均与相应阳性药物(PXR/Rif、FXR/CDCA和LXRα/T0901317)呈剂量依赖性增长;4仅PXR激动剂Rif、FXR激动剂CDCA和LXRα激动剂T0901317可分别明显增加相应筛选模型的报告基因活性,分别重复5次试验后,计算得Z'值分别为0.58、0.66和0.63。结论该研究建立的PXR、FXR和LXRα激动剂高通量筛选模型,具有良好的特异性和稳定性,适用于对PXR、FXR和LXRα受体激动剂的筛选,进而开发以核受体作为药物靶点的药物。Aim To develop an in vitro high throughput drug screening system based on reporter gene assay for identification of novel compounds with PXR,FXR and LXRα agonist activity.Methods The expressions of exogenous PXR,FXR and LXRαgene in HEK293,Hep G2 and LS174 T cells were examined by Real-Time quantity PCR. p SG5-h PXR and p GL3-XREMCYP3A4,p EGFP-N3-h FXR and EcRE-TK-Luc,p CMX-FLAGh LXRα and p GL3-XREM-CYP3A4 were cotransfected into cells and the optimal ratio of three plasmids was determined. The dose-response relationship between the positive drug and the fold induction was determined. The specificity of the model was examined,and the repeatability was also determined by Z' value.Results 1 The PXR,FXR and LXRα mRNA expression in HEK293 cell is low among three different cells. 2 reporter gene vector and expression plasmid ratio of 1 ∶ 1,2 ∶ 1 and 2 ∶ 1 were proved to be suitable for highest relative luciferase activity for PXR,FXR or LXRα agonist screening model. 3 The relative luciferase activity was induced by Rif,CDCA or T0901317 in a dose-dependent manner. 4 Only Rif,CDCA or T0901317 could significantly increase the relative luciferase activity in PXR,FXR or LXRα agonist screening model,no effect of other nuclear receptors agonist was observed,and the values of Z'-factor for PXR,FXR and LXRα agonist screening model were 0. 58,0. 66 and 0. 63,respectively. Conclusion An in vitro PXR,FXR and LXRα agonist high-throughput screening models are developed with acceptable specificity and repeatability,and the models can be used to screen PXR,FXR and LXRα agonist.

关 键 词：核受体 报告基因 高通量筛选 PXR FXR LXRΑ 

分 类 号：R392.11[医药卫生—免疫学] R394.2[医药卫生—基础医学]