Drewlo Sascha, Baczyk Dora, Dunk Caroline, Kingdom John
Womens and Infants Health, Samuel Lunenfeld Research Institute, Department of Obstetrics and Gynaecology, Mount Sinai Hospital, University of Toronto, Ontario, Canada.
Methods Mol Biol. 2008;475:363-82. doi: 10.1007/978-1-59745-250-2_21.
A healthy syncytium in the placenta is vital to a successful pregnancy. The trophoblast builds up the natural barrier between the mother and the developing fetus and is the site of gas, nutrition, and waste exchange. An inadequate formation of this tissue leads to several pathologies of pregnancy, which may result in fetal death during the second trimester or iatrogenic preterm delivery due to intrauterine growth restriction, preeclampsia, or abruption.Cytotrophoblastic cells fuse constantly with the overlying syncytiotrophoblast/syncytium to maintain the function of the trophoblast. Syncytin-1 is the only molecule known to directly induce fusion in the placental trophoblast. Many other proteins, such as gap junctions (e.g., connexin 40) and transcription factors, play a role in the molecular pathways directing the trophoblast turn over. Despite the significance of this process for successful placentation, the mechanisms regulating its activity remain poorly understood.In this chapter we present several different model systems that can be utilized to investigate the regulation of the cell fusion process in the trophoblast. We describe cell-based assays as well as tissue-related protocols. We show how fusion can be monitored in (1) BeWo cells as a trophoblast cell line model, (2) HEK239 using syncytin-1 as a fusion molecule, and (3) a floating villi explant model. Furthermore, we will present strategies to inhibit fusion in the different models. These techniques represent powerful tools to study the molecular mediators of cell fusion in the trophoblast.
胎盘内健康的合体滋养层对成功妊娠至关重要。滋养层细胞构建了母体与发育中胎儿之间的天然屏障，也是气体、营养物质和废物交换的场所。该组织形成不足会导致多种妊娠病理情况，可能导致孕中期胎儿死亡，或因子宫内生长受限、先兆子痫或胎盘早剥而导致医源性早产。细胞滋养层细胞不断与覆盖其上的合体滋养层/合体融合，以维持滋养层的功能。Syncytin-1是已知唯一能直接诱导胎盘滋养层细胞融合的分子。许多其他蛋白质，如间隙连接（如连接蛋白40）和转录因子，在指导滋养层更新的分子途径中发挥作用。尽管这一过程对成功胎盘形成具有重要意义，但其活性的调节机制仍知之甚少。在本章中，我们介绍了几种不同的模型系统，可用于研究滋养层细胞融合过程的调节。我们描述了基于细胞的检测方法以及与组织相关的实验方案。我们展示了如何在以下模型中监测融合情况：（1）作为滋养层细胞系模型的BeWo细胞；（2）使用Syncytin-1作为融合分子的HEK293细胞；（3）漂浮绒毛外植体模型。此外，我们还将介绍在不同模型中抑制融合的策略。这些技术是研究滋养层细胞融合分子介质的有力工具。
