【摘要】：目的建立基于報告基因法的高通量篩選細胞模型,用來發(fā)現(xiàn)PXR、FXR和LXRα受體激動劑。方法利用Real-time定量PCR方法比較HEK293、Hep G2和LS174T細胞中內(nèi)源性核受體PXR、FXR和LXRα的表達量,將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等質(zhì)粒分別共轉染到工具細胞中,優(yōu)化共轉染比例,并考察陽性藥與螢光素酶報告基因表達強度的量效關系、模型特異性和穩(wěn)定性。結果 1根據(jù)Real-time定量PCR結果,模型選用低表達PXR、FXR和LXRα的HEK293細胞作為工具細胞;2根據(jù)不同共轉染比例對報告基因活性的結果,PXR、FXR和LXRα報告基因藥物篩選模型的報告基因和過表達質(zhì)粒比例,最終分別選擇1∶1、2∶1和2∶1;3模型中,報告基因活性均與相應陽性藥物(PXR/Rif、FXR/CDCA和LXRα/T0901317)呈劑量依賴性增長;4僅PXR激動劑Rif、FXR激動劑CDCA和LXRα激動劑T0901317可分別明顯增加相應篩選模型的報告基因活性,分別重復5次試驗后,計算得Z'值分別為0.58、0.66和0.63。結論該研究建立的PXR、FXR和LXRα激動劑高通量篩選模型,具有良好的特異性和穩(wěn)定性,適用于對PXR、FXR和LXRα受體激動劑的篩選,進而開發(fā)以核受體作為藥物靶點的藥物。
[Abstract]:Objective to establish a high throughput screening cell model based on reporter gene method for the detection of PXR,FXR and LXR 偽 receptor agonists. Methods Real-time quantitative PCR was used to compare the expression of endogenous nuclear receptors PXR,FXR and LXR 偽 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 偽 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 偽 were selected as tool cells, (2) the ratio of reporter gene and overexpression plasmid of PXR,FXR and LXR 偽 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 偽 reporter gene screening model, and the ratio of reporter gene and overexpression plasmid of PXR,FXR and LXR 偽 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 偽 / T0901317). 4 only PXR agonist Rif,FXR agonist CDCA and LXR 偽 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 偽 agonists established by this study has good specificity and stability, and is suitable for screening PXR,FXR and LXR 偽 receptor agonists, and then develop drugs with nuclear receptors as drug targets.
【作者單位】： 中山大學藥學院藥物代謝與藥動學實驗室;
【基金】：廣東省新藥設計與評價重點實驗室開放基金(No 2011A060901014-009)
【分類號】：R96

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