【摘要】 目的构建一种新的、具有生物活性检测能力的人过氧化物酶体增殖物激活受体α(hPPARα)配体药物筛选细胞模型。方法采用Lipofectamine 2000将含人PPARα基因质粒phPPARα-IRES2-EGFP、含人PPRE报告质粒ptk-PPRE×3-luc及内部对照质粒pRL-CMV共转染293T细胞,并对phPPARα-IRES2-EGFP转染效率进行流式细胞仪(FCM)分析;通过双荧光素酶报告基因法(DLR)检测不同浓度、不同时间阳性药物WY14643干预共转染细胞体系的荧光素酶表达活性;实验还换用RXRα激动剂全反式维甲酸(ATRA)干预,以评价该细胞模型反应的特异性;并用贝特类降脂药苯扎贝特、环丙贝特、氯贝丁酯对建立的药物筛选细胞模型进行应用能力评价。结果 FCM检测共转染293T细胞phPPARα-IRES2-EGFP质粒转染效率为68%;DLR检测WY14643干预该hPPARα药物筛选模型获得了理想的量-效、时-效关系结果,且该模型不能通过RXRα配体激动剂ATRA激活。苯扎贝特、环丙贝特、氯贝丁酯干预模型均呈现良好的量-效反应性,且反应强度存在差异(P<0.05)。结论成功构建了基于hPPARα为靶标的药物筛选细胞模型,为筛选具有生物活性的hPPARα配体激动剂新药提供了一种可靠的新平台。更多还原

【Abstract】 OBJECTIVE To establish a novel cell-based model for the screening of human PPARα agonist ligands.METHODS Recombinant plasmid phPPARα-IRES2-EGFP,firefly luciferase-containing report plasmid ptk-PPRE×3-luc and renilla luciferase-containing inner control plasmid pRL-CMV were co-transfected into 293T cells using lipofectamine 2000.The transfection efficiency of phPPARα-IRES2-EGFP was measured by flow cytometry(FCM).Dual-luciferase reporter assay(DLR) was used to detect the luciferase activity of co-transfected 293T cells under concentration gradient at different time points of positive drug WY14643 intervention.Furthermore,WY14643 was replaced by all-trans-retinoic acid(ATRA),a RXRα specific agonist,for the purpose of clarifying the specificity of the cell model.Moreover,fibrates such as bezafibrate,ciprofibrate and clofibrate,which are the potent clinical hypolipidemic agents,were used to verify the utilization ability of the cell model.RESULTS FCM analysis revealed that the transfection efficiency of phPPARα-IRES2-EGFP in co-transfected 293T cells was 68%.DLR detected ideal dose-response and time-course relationships for WY14643 intervention whereas the cell model could not activated by ATRA.The cell model responded ideally to fibrates(bezafibrate,ciprofibrate,and clofibrate) as expected but the response intensities were different(P<0.05).CONCLUSION The 293T cell-based drug screening model targeting specific nuclear receptor of hPPARα has been established here and is available for the drug-screening of unknown hPPARα agonist ligands.更多还原