PPARγ激动剂筛选模型的建立及其应用

Establishment and Application of a Cell Model for Screening PPARγ Agonists

目的建立一种简便实用的细胞模型,用于筛选过氧化物酶增殖剂激活受体γ(PPARγ)的新配体激动剂。方法在U937细胞中,共转染PPARγ表达和报告质粒,构建PPARγ激动剂筛选模型,单独转染PPARγ报告质粒作为阴性对照。转染细胞中加入候选药物,24h后裂解细胞,测定细胞内报告质粒所表达虫荧光素酶的活性,该活性大小即代表药物激动PPARγ的能力。结果已知的PPARγ配体激动剂吡格列酮使虫荧光素酶活性增强了4.48倍(P<0.001),验证了模型的有效性。高密度脂蛋白(HDL)不能激动PPARγ,而氧化后随剂量增加,使虫荧光素酶活性增强1.33倍(25μg/mL)和1.78倍(50μg/mL),但没有达到统计学差异(P=0.058,P=0.054);白藜芦醇使虫荧光素酶强度增加了1.78倍和2.47倍(P=0.033,P=0.01)。结论通过细胞共转染PPARγ表达质粒和编码虫荧光素酶的报告质粒,可以建立简便的PPARγ激动剂筛选模型。HDL氧化后可能会产生激动PPARγ的成份,而白藜芦醇具有较强的PPARγ激动能力。

Objective To establish a cell model for quick screening new peroxisome proliferator-activated receptors γ （ PPAR γ） agonists and utilize it to analyze the capability of high-density lipoprotein （ HDL）, oxidized highdensity lipoprotein （ox-HDL） and resveratrol in activating PPARγ. Methods U937 cells were co-transfected with PPAR γ expression plasmids and PPAR γ reporter plasmids. Cells transfected with reporter plasmids alone served as negative controls. Transfected cells were cultured with known PPAR γ agonist pioglitazone, ox-HDL and resveratrol for 24 hours, then lysated and assayed for reporter luciferase activity, which represented the compounds＇ capability to activate PPAR γ. Results Pioglitazone significantly activated PPARγ, bringing luciferase activity up to 4.48 folds compared with the control （P 〈0. 001 ）. HDL didn＇t change luciferase activity, while ox-HDL had a trend to increase luciferase activity by 1.33 （25 μg/mL） and 1. 78 folds （50 μg/mL ）, but didn＇t meet statistical significance （P =0.058 and 0. 054, respectively）. Resveratrol dose-dependently boosted luciferase activity by 1. 78 （P = 0. 033） and 2.47 folds（P =0.01 ）. All compounds had no influence on luciferase activity in negative control cells. Conclusion We successfully established a cell model for screening potential PPAR γ agonists and found that oxidi- zation of HDL might create agonists of PPAR γ. Resveratrol was most likely to be an agonist of PPAR γ.