CAJ | 학술논문

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Re-al-time定量PCR方法比较HEK293、HepG2和LS174T细胞中内源性核受体 PXR、FXR 和 LXRα的表达量,将 pSG5-hPXR 和 pGL3-XREM-CYP3A4、pEGFP-N3-hFXR 和 EcRE-TK-Luc、 pCMX-FLAG-hLXRα和 pGL3-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。
목적：건립기우보고기인법적고통량사선세포모형,용래발현PXR、FXR화LXRα수체격동제。방법이용Re-al-time정량PCR방법비교HEK293、HepG2화LS174T세포중내원성핵수체 PXR、FXR 화 LXRα적표체량,장 pSG5-hPXR 화 pGL3-XREM-CYP3A4、pEGFP-N3-hFXR 화 EcRE-TK-Luc、 pCMX-FLAG-hLXRα화 pGL3-XREM-CYP3A4등질립분별공전염도공구세포중,우화공전염비례,병고찰양성약여형광소매보고기인표체강도적량효관계、모형특이성화은정성。결과①근거Real-time정량PCR결과,모형선용저표체PXR、FXR화LXRα적HEK293세포작위공구세포；②근거불동공전염비례대보고기인활성적결과,PXR、FXR화LXRα보고기인약물사선모형적보고기인화과표체질립비례,최종분별선택1∶1、2∶1화2∶1；③모형중,보고기인활성균여상응양성약물( PXR/Rif、FXR/CDCA화LXRα/T0901317)정제량의뢰성증장；④부 PXR 격동제 Rif、FXR격동제CDCA화LXRα격동제T0901317가분별명현증가상응사선모형적보고기인활성,분별중복5차시험후,계산득Z′치분별위0．58、0．66화0．63。결론해연구건립적PXR、FXR화LXRα격동제고통량사선모형,구유량호적특이성화은정성,괄용우대PXR、FXR화LXRα수체격동제적사선,진이개발이핵수체작위약물파점적약물。
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, HepG2 and LS174T cells were examined by Real-Time quantity PCR. pSG5-hPXR and pGL3-XREM-CYP3A4, pEGFP-N3-hFXR and EcRE-TK-Luc, pCMX-FLAG-hLXRα and pGL3-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 ex-amined, 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. ②reporter 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. ④Only Rif, CDCA or T0901317 could significantly increase the relative luciferase activity in PXR,FXR or LXRα agonist screening model, no effect of other nuclear re-ceptors agonist was observed, and the values of Z′-factor for PXR, FXR and LXRαagonist 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 devel-oped with acceptable specificity and repeatability, and the mod-els can be used to screen PXR, FXR and LXRα agonist.