基于报告基因检测的β_3肾上腺素受体激动剂筛选模型的建立与应用

Establishment and application of a screening model for β_3 adrenoceptor agonists based on reporter gene assay

目的:建立基于双荧光素酶报告基因检测的β3肾上腺素受体(β3-AR)激动剂筛选模型,并用于β3-AR激动剂的筛选。方法:应用免疫细胞化学法鉴定β3-AR在β3-CHO细胞上的稳定表达,并将pCRE-luc质粒与pRL-TK质粒共同瞬时转染β3-CHO细胞,建立一种基于双荧光素酶报告基因检测的β3-AR激动剂筛选模型。通过检测报告基因萤火虫荧光素酶与海肾荧光素酶活性的比值,来间接反映配体对β3-AR的激动活性。并以β-AR激动剂异丙肾上腺素刺激对模型的有效性进行验证,在此基础上以此模型对20种新化合物的β3-AR激动活性进行筛选。结果:β3-CHO细胞经免疫细胞化学法鉴定有特异性受体蛋白表达。通过将两个报告基因质粒转染该细胞后,与加入溶剂对照相比,加入异丙肾上腺素可显著促进荧光素酶报告基因的表达,表明该模型有效、可靠。以此模型从20个化合物中初筛出8个活性较强的β3-AR激动剂。结论:本研究建立了基于双荧光素酶报告基因检测的β3-AR激动剂筛选模型,并以此模型筛选发现了几个活性较强的β3-AR激动剂。

Objective： To establish a screening model for β3 adrenergic receptor（β3-AR） agonists based on dual-luciferase reporter gene assay and apply it to screening of β3-AR agonists.Methods： The expression of β3-AR on β3-CHO cells was identified by immunocytochemistry method.A screening model of β3-AR agonists based on dual-luciferase reporter gene assay was established by β3-CHO cells cotransfected with pCRE-luc and pRL-TK plasmids.The activities of firefly and renilla luciferases were measured independently in each sample,and the agonist activities of compounds to β3-AR were determined by the quotient of the two activities.The reliability of the cell model was evaluated by stimulation of isoprenaline,a β-AR agonist.The agonist activities of 20 new compounds were tested by this model.Results：The reliability of the model was demonstrated by the results that the expression of luciferase reporter genes were enhanced by isoprenaline compared with solvent control.Eight compounds with high agonist activity to β3-AR were screened out from 20 new compounds.Conclusion： A screening model for β3-AR agonists based on dual-luciferase reporter gene assay is established successfully,and a few active agonists for β3-AR have been discovered.