Institute of Experimental and Clinical Pharmacology and Toxicology, Department of Toxicology, University of Tübingen, Tübingen, Germany.
Reprod Toxicol. 2010 Aug;30(1):103-12. doi: 10.1016/j.reprotox.2009.12.002. Epub 2009 Dec 14.
A stem cell-based reporter assay was developed to detect drug-induced alterations in the canonical Wnt/beta-catenin signaling pathway, which is involved in the regulation of early embryonic development. The so-called ReProGlo assay allows simultaneous determination of cell viability and luciferase reporter activity in a high throughput 96-well microtiter format. A clone of mouse embryonic stem (mES) cells stably expressing the SuperTopFlash reporter was established. This allows Wnt pathway activity determinations in undifferentiated mES cells and their differentiated descendants. Several test chemicals were analyzed in the new assay system. Known embryotoxicants like retinoic acid or lithium chloride induced concentration-dependent increases in reporter activity. The potency of valproic acid and a series of structural analogs to activate the Wnt pathway correlated well with their reported teratogenic activity in the mouse. Cyclophosphamide was also active but only after metabolic activation by hepatocytes. The new test may help to predict embryotoxic potential of chemicals.
建立了一种基于干细胞的报告基因检测方法，用于检测药物诱导的经典 Wnt/β-连环蛋白信号通路改变，该通路参与调节早期胚胎发育。所谓的 ReProGlo 检测法允许在高通量 96 孔微量滴定板格式中同时测定细胞活力和荧光素酶报告基因活性。建立了稳定表达 SuperTopFlash 报告基因的小鼠胚胎干细胞 (mES) 细胞克隆。这使得可以在未分化的 mES 细胞及其分化的后代中测定 Wnt 途径活性。在新的检测系统中分析了几种测试化学品。已知的胚胎毒性物质，如维甲酸或氯化锂，会诱导报告基因活性的浓度依赖性增加。丙戊酸和一系列结构类似物激活 Wnt 途径的效力与其在小鼠中的致畸活性密切相关。环磷酰胺也具有活性，但只有在肝细胞代谢激活后才具有活性。新的测试方法可能有助于预测化学品的胚胎毒性潜力。
