Bremer S, van Dooren M, Paparella M, Kossolov E, Fleischmann B, Hescheler J
ECVAM, Institute for Health and Consumer Protection, Joint Research Centre, European Commission, 21020 Ispra, Italy.
University of Cologne, Department of Neurophysiology, Robert-Koch-Strasse 39, 50931 Cologne, Germany.
Altern Lab Anim. 1999 May-Jun;27(3):471-84. doi: 10.1177/026119299902700303.
Transgenic embryonic stem cells were used to determine the embryotoxic effects of chemicals on the development of embryonic tissues. This investigation supports an ongoing validation study, aimed at reducing the time-consuming procedure currently in use, and at providing more-objective and more-detailed information on the embryotoxic potentials of chemicals. Green fluorescence protein (GFP) was used as a reporter gene and was linked to a human α-cardiac-specific promoter. The expression of GFP was switched on after specific activation of the human α-actin promoter. This permitted the easy quantification of cardiac cells by using a fluorescence-activated cell sorter (FACS). The percentage of cardiac precursor cells was calculated from the FACS-distribution pattern of cells which fluoresced versus the total number of cells. The percentage of cardiac precursor cells increased from 25% in embryoid bodies on day 3, to 86% on day 7. However, in 11-day-old embryoid bodies, the percentage decreased to 35%. Five chemicals with known embryotoxic potentials were compared with respect to the IC50 (concentration causing 50% inhibition of measured effect) values obtained by various in vitro endpoints (for example, cytotoxicity, morphology). The results showed a higher sensitivity of endpoints used for the analysis of specific effects on the selected target tissue. The data also showed the need to develop in vitro methods with specific endpoints which account for the complexity of embryotoxicology.
转基因胚胎干细胞被用于确定化学物质对胚胎组织发育的胚胎毒性作用。这项研究支持了一项正在进行的验证研究，旨在减少目前使用的耗时程序，并提供关于化学物质胚胎毒性潜力的更客观、更详细的信息。绿色荧光蛋白（GFP）被用作报告基因，并与人类α-心脏特异性启动子相连。在人类α-肌动蛋白启动子特异性激活后，GFP的表达被开启。这使得通过使用荧光激活细胞分选仪（FACS）能够轻松定量心脏细胞。心脏前体细胞的百分比是根据发出荧光的细胞的FACS分布模式与细胞总数计算得出的。心脏前体细胞的百分比从第3天胚状体中的25%增加到第7天的86%。然而，在11天大的胚状体中，该百分比降至35%。将五种具有已知胚胎毒性潜力的化学物质就通过各种体外终点（例如细胞毒性、形态学）获得的IC50（导致所测效应50%抑制的浓度）值进行了比较。结果表明，用于分析对选定靶组织的特定效应的终点具有更高的敏感性。数据还表明需要开发具有特定终点的体外方法，以考虑胚胎毒理学的复杂性。
