PPARγ激动剂体外筛选模型的建立

Development of an in vitro model for PPARγ agonists screening

目的:构建一种过氧化物酶体增殖物激活受体(peroxisome proliferator-activated receptor,PPAR)γ激动剂体外筛选模型,为筛选具有潜在治疗效果的PPARγ激动剂提供研究手段。方法:用脂质体2000(LipofectamineTM2000)将含有PPARγ基因质粒(pIRES2-PPARγ)、萤火虫荧光素酶报告基因质粒(pPPRE×3-TK-Luciferase)以及内参照质粒(pRL-TK)按不同比例共转染入人胚胎肾细胞HEK293细胞,并对3种质粒比例进行优化;以不同配体处理转染3种质粒的HEK293细胞,通过检测荧光素酶相对活性来确定加入配体对PPARγ的激动效应;采用经典的PPARγ激动剂和PPARγ特异性拮抗剂,其他核受体激动剂以及PPARα激动剂考察不同化合物对PPARγ的激动作用程度,确定模型的特异性;以Z’值考察模型重复性;并观察了PPARγ激动剂的作用时间特点。结果:PPARγ质粒、报告基因质粒以及内参照质粒比例为2∶6∶1时相对荧光素酶活性最高;PPARγ激动剂罗格列酮可显著增加相对荧光素酶的活性而PPARγ特异性拮抗剂GW9662可抑制这种作用,且相对荧光素酶的活性随着处理时间的增加而升高;地塞米松,全反式维甲酸,雌二醇和非诺贝特无上述活性;重复9次实验后计算得Z’=0.70。结论:本研究成功建立了一种PPARγ激动剂筛选模型,且模型特异性和重复性较好,为进一步筛选具有PPARγ激动剂提供了理想的研究手段。

Objective：To develop an in vitro peroxisome proliferator-activated receptor（PPAR）γ ligand screening system for identifying novel compounds with PPARγ agonist activity.Methods：Human PPARγ plasmid（pIRES2-PPARγ）,firefly luciferase reporter plasmid（pPPRE×3-TK-Luciferase） and an internal reference plasmid（pRL-TK）were cotransfected into HEK293 cells by using LipofectamineTM 2000,and the optimal ratio of three plasmids was determined.Luciferase expression intensity was determined to assess the PPARγ agonist activity after the potential ligand being added to cotransfected HEK293 cells.Specificity of the model was examined by treatment of PPARγ agonist and PPARγ specific antagonist and several nuclear receptor agonists and PPARα agonist;repeatability was also determined by Z＇ value and time characteristics of PPAR agonist action was observed.Results：PPARγ plasmid,reporter gene vector and internal control plasmid ratio of 2∶6∶1 was proved to be suitable for highest relative luciferase activity.PPARγ agonist rosiglitazone can significantly increase the relative luciferase activity and co-treatment with PPARγ specific antagonist GW9662 resulted in significantly reduced expressions of luciferase and increased relative luciferase activity in the HEK293 cells in a time dependent manner.Dexamethasone,all-trans retinoic acid,17β-estradiol and fenobrate did not alter the relative luciferase activity.Value of Z＇ was 0.70 after repeating experiments for 9 times.Conclusions：An in vitro PPARγ agonist screening system is developed with acceptable specificity and repeatability.The model can be used for PPARγ partial agonist screening from synthetic or natural compounds.