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
发布时间：2019-06-21 04:34
【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。
[Abstract]:Objective to establish a high throughput screening cell model based on reporter gene method for the detection of PXR,FXR and LXR 伪 receptor agonists. Methods Real-time quantitative PCR was used to compare the expression of endogenous nuclear receptors PXR,FXR and LXR 伪 in HEK293,Hep G2 and LS174T cells. PSG5-h PXR and pGL3-XREM-CYP3A4,p EGFP-N3-h FXR and EcRETK-Luc,p CMX-FLAG-h LXR 伪 and pGL3-XREM-CYP3A4 were co-transfected into tool cells, respectively. The co-transfection ratio was optimized, and the dose-effect relationship between positive drugs and luciferase reporter gene expression intensity was investigated. Model specificity and stability. Results (1) according to the results of Real-time quantitative PCR, HEK293 cells with low expression of PXR,FXR and LXR 伪 were selected as tool cells, (2) the ratio of reporter gene and overexpression plasmid of PXR,FXR and LXR 伪 reporter gene drug screening model was 1: 1, 2: 1 and 2: 1, respectively, according to the results of reporter gene activity of PXR,FXR and LXR 伪 reporter gene screening model, and the ratio of reporter gene and overexpression plasmid of PXR,FXR and LXR 伪 reporter gene drug screening model was 1: 1, 2: 1 and 2: 1, respectively. 3 in the model, the reporter gene activity increased in a dose-dependent manner with the corresponding positive drugs (PXR/Rif,FXR/CDCA and LXR 伪 / T0901317). 4 only PXR agonist Rif,FXR agonist CDCA and LXR 伪 agonist T0901317 significantly increased the reporter gene activity of the corresponding screening model, and the calculated Z' values were 0.580.66 and 0.63, respectively. Conclusion the high throughput screening model of PXR,FXR and LXR 伪 agonists established by this study has good specificity and stability, and is suitable for screening PXR,FXR and LXR 伪 receptor agonists, and then develop drugs with nuclear receptors as drug targets.
【作者单位】： 中山大学药学院药物代谢与药动学实验室;
【基金】：广东省新药设计与评价重点实验室开放基金(No 2011A060901014-009)
【分类号】：R96
[Abstract]:Objective to establish a high throughput screening cell model based on reporter gene method for the detection of PXR,FXR and LXR 伪 receptor agonists. Methods Real-time quantitative PCR was used to compare the expression of endogenous nuclear receptors PXR,FXR and LXR 伪 in HEK293,Hep G2 and LS174T cells. PSG5-h PXR and pGL3-XREM-CYP3A4,p EGFP-N3-h FXR and EcRETK-Luc,p CMX-FLAG-h LXR 伪 and pGL3-XREM-CYP3A4 were co-transfected into tool cells, respectively. The co-transfection ratio was optimized, and the dose-effect relationship between positive drugs and luciferase reporter gene expression intensity was investigated. Model specificity and stability. Results (1) according to the results of Real-time quantitative PCR, HEK293 cells with low expression of PXR,FXR and LXR 伪 were selected as tool cells, (2) the ratio of reporter gene and overexpression plasmid of PXR,FXR and LXR 伪 reporter gene drug screening model was 1: 1, 2: 1 and 2: 1, respectively, according to the results of reporter gene activity of PXR,FXR and LXR 伪 reporter gene screening model, and the ratio of reporter gene and overexpression plasmid of PXR,FXR and LXR 伪 reporter gene drug screening model was 1: 1, 2: 1 and 2: 1, respectively. 3 in the model, the reporter gene activity increased in a dose-dependent manner with the corresponding positive drugs (PXR/Rif,FXR/CDCA and LXR 伪 / T0901317). 4 only PXR agonist Rif,FXR agonist CDCA and LXR 伪 agonist T0901317 significantly increased the reporter gene activity of the corresponding screening model, and the calculated Z' values were 0.580.66 and 0.63, respectively. Conclusion the high throughput screening model of PXR,FXR and LXR 伪 agonists established by this study has good specificity and stability, and is suitable for screening PXR,FXR and LXR 伪 receptor agonists, and then develop drugs with nuclear receptors as drug targets.
【作者单位】： 中山大学药学院药物代谢与药动学实验室;
【基金】：广东省新药设计与评价重点实验室开放基金(No 2011A060901014-009)
【分类号】：R96
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