基于人PPARα为靶标的药物筛选细胞模型的建立与应用评价  

A Cell-Based Screening Model for Human PPARα Agonist Ligands Discovery and Its Applicability

作　　者：马晶晶[1] 张潜[2] 方宁[1] 万卫红[1] 刘祖林[1] 章涛[1] 

机构地区：[1]遵义医学院附属医院贵州省细胞工程重点实验室,贵州遵义563003 [2]遵义医学院人体解剖学教研室,贵州遵义563003

出　　处：《中国药学杂志》2011年第23期1798-1804,共7页Chinese Pharmaceutical Journal

基　　金：贵州省科学技术基金项目(黔科合J字2008-2152号)

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。OBJECTIVE To establish a novel cell-based model for the screening of human PPARct agonist ligands. METHODS Recombinant plasmid phPPARα-IRES2-EGFP, firefly luciferase-containing report plasmid ptk-PPRE × 3-1uc 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 RXRct 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 hypolipi- demic agents, were used to verify the utilization ability of the cell model. RESULTS FCM analysis revealed that the transfection effi- ciency of phPPARc^-IRES2-EGFP in co-transfected 293T ceils was 68%. DLR detected ideal dose-response and time-course relation- ships 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.

关 键 词：过氧化物酶体增殖物激活受体Α 药物筛选 293T细胞 报告基因 

分 类 号：R965.1[医药卫生—药理学]