Sagrillo-Fagundes Lucas, Clabault Hélène, Laurent Laetitia, Hudon-Thibeault Andrée-Anne, Salustiano Eugênia Maria Assunção, Fortier Marlène, Bienvenue-Pariseault Josianne, Wong Yen Philippe, Sanderson J Thomas, Vaillancourt Cathy
INRS-Institut Armand-Frappier.
INRS-Institut Armand-Frappier;
J Vis Exp. 2016 Jul 30(113):54228. doi: 10.3791/54228.
This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density centrifugation, media gradient isolation and immunomagnetic purification. As observed in vivo, mononucleated villous cytotrophoblast cells in primary culture differentiate into multinucleated syncytiotrophoblast cells after 72 hr. Compared to normoxia (8% O2), villous cytotrophoblast cells that undergo hypoxia/reoxygenation (0.5% / 8% O2) undergo increased oxidative stress and intrinsic apoptosis, similar to that observed in vivo in pregnancy complications such as preeclampsia, preterm birth, and intrauterine growth restriction. In this context, primary villous trophoblasts cultured under hypoxia/reoxygenation conditions represent a unique experimental system to better understand the mechanisms and signalling pathways that are altered in human placenta and facilitate the search for effective drugs that protect against certain pregnancy disorders. Human villous trophoblasts produce melatonin and express its synthesizing enzymes and receptors. Melatonin has been suggested as a treatment for preeclampsia and intrauterine growth restriction because of its protective antioxidant effects. In the primary villous cytotrophoblast cell model described in this paper, melatonin has no effect on trophoblast cells in normoxic state but restores the redox balance of syncytiotrophoblast cells disrupted by hypoxia/reoxygenation. Thus, human villous trophoblast cells in primary culture are an excellent approach to study the mechanisms behind the protective effects of melatonin on placental function during hypoxia/reoxygenation.
本方案描述了如何通过连续酶消化从足月胎盘中分离绒毛细胞滋养层细胞，随后进行密度离心、介质梯度分离和免疫磁珠纯化。如在体内观察到的那样，原代培养的单核绒毛细胞滋养层细胞在72小时后分化为多核合体滋养层细胞。与常氧（8% O₂）相比，经历缺氧/复氧（0.5% / 8% O₂）的绒毛细胞滋养层细胞会经历氧化应激增加和内在凋亡，类似于在子痫前期、早产和宫内生长受限等妊娠并发症的体内观察结果。在此背景下，在缺氧/复氧条件下培养的原代绒毛滋养层细胞代表了一个独特的实验系统，以更好地理解人类胎盘中改变的机制和信号通路，并有助于寻找预防某些妊娠疾病的有效药物。人绒毛滋养层细胞产生褪黑素并表达其合成酶和受体。由于其保护性抗氧化作用，褪黑素已被建议用于治疗子痫前期和宫内生长受限。在本文所述的原代绒毛细胞滋养层细胞模型中，褪黑素对常氧状态下的滋养层细胞没有影响，但可恢复因缺氧/复氧而破坏的合体滋养层细胞的氧化还原平衡。因此，原代培养的人绒毛滋养层细胞是研究褪黑素在缺氧/复氧期间对胎盘功能保护作用背后机制的极佳方法。
