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首页> 外文期刊>Environmental toxicology and chemistry >A TELEOST IN VITRO REPORTER GENE ASSAY TO SCREEN FOR AGONISTS OF THE PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS
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A TELEOST IN VITRO REPORTER GENE ASSAY TO SCREEN FOR AGONISTS OF THE PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS

机译：体外报告基因基因的最新研究，可筛选过氧化物酶增殖物激活受体的激动剂

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摘要

Several contaminants detected in aquatic ecosystems are agonists of peroxisome proliferator-activated receptors (PPARs). Peroxisome proliferator-activated receptors interact with the retinoid X receptor (RXR) to activate the transcription of genes that control a variety of physiological functions. We cloned and sequenced partial cDNA fragments of rainbow trout (Oncorhynchus mykiss) PPARa and PPARβ from rainbow trout (rt) gill-W1 cells, a cell line derived from rainbow trout gills; predicted amino acid identities are 77% and 82% compared with their respective human homologs and 83 to 88% and 91 to 98% identical to fish homologs. A reporter gene assay was developed by transfecting rt-gill-W1 cells with a reporter gene construct containing the peroxisome proliferator response element (PPRE) of the rat liver 3-ketoacyl-CoA thiolase B (TB) gene, which drives luciferase expression. Agonists of both PPARa (WY14,643 and gemfibrozil) and PPARβ (bezafibrate) induced luciferase activity, while rosiglitazone, a PPARγ agonist, was not effective. The fibrate drug, bezafibrate increased luciferase activity in a dose-dependent manner, but addition of 50 nM 9-cis-retinoic acid to the transfected rt-gill-W1 cell culture maximized the sensitivity of the assay so that bezafibrate could be detected at concentrations as low as 6 nM. Extracts from treated domestic wastewater containing fibrate drugs induced luciferase activity in the transfected gill cells. This in vitro reporter gene assay shows promise as a rapid and sensitive technique for screening environmental samples for PPAR-active substances.
机译：在水生生态系统中检测到的几种污染物是过氧化物酶体增殖物激活受体（PPAR）的激动剂。过氧化物酶体增殖物激活的受体与类维生素A X受体（RXR）相互作用，以激活控制多种生理功能的基因的转录。我们从虹鳟鱼rt细胞W1细胞（虹鳟鱼g细胞）中克隆了虹鳟鱼PPARa和PPARβ的部分cDNA片段并进行了测序。与它们各自的人类同源物相比，预测的氨基酸同一性分别为77％和82％，与鱼类同源物的同源性为83至88％和91至98％。通过用报告基因构建体转染rt-gill-W1细胞，开发了一种报告基因测定法，该报告基因构建体包含驱动肝脏萤光素酶表达的大鼠肝脏3-酮酰基-CoA硫解酶B（TB）基因的过氧化物酶体增殖物应答元件（PPRE）。 PPARa（WY14,643和吉非贝齐）和PPARβ（苯扎贝特）的激动剂均可诱导萤光素酶活性，而PPARγ激动剂罗格列酮则无效。纤维状药物苯扎贝特以剂量依赖性方式增加了荧光素酶的活性，但向转染的rt-gill-W1细胞培养物中添加50 nM 9-顺-视黄酸可最大程度地提高测定的灵敏度，因此可以在一定浓度下检测到苯扎贝特低至6 nM。处理过的含有贝特类药物的生活污水中的提取物在转染的ill细胞中诱导了萤光素酶活性。这种体外报道基因测定法显示出作为一种快速灵敏的技术来筛选环境样品中PPAR活性物质的前景。

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