Department of Anatomy, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, 30173 Hannover, Germany.
Placenta. 2010 Jan;31(1):67-74. doi: 10.1016/j.placenta.2009.10.011. Epub 2009 Nov 14.
In the bovine placenta, multinucleate trophoblast giant cells (TGC), evolving from uninucleate trophoblast cells, are crucial for feto-maternal interaction as they show endocrine activity and the ability to migrate and fuse with caruncular epithelial cells. In contrast to caruncular epithelial cells, the isolation and culture of bovine trophoblast cells is complicated because they cease to express their specific products, like placental lactogen (PL), during prolonged culture. In the present study, we aimed to establish a bovine cotyledonary trophoblast cell line targeting our long term goal to develop an in vitro model for the bovine placenta. Therefore, the functional activity of important signalling pathways was tested. Primary trophoblast cells were isolated from a bovine cotyledon of a male fetus and successfully subcultured and cryopreserved. The obtained cell line, termed F3, showed epithelial morphology and characteristic binuclear giant cells in small numbers through all passages. The trophoblastic origin of F3 cells was verified by amplification of a Y-chromosome specific DNA-sequence and the presence of PL mRNA. Immunofluorescence demonstrated that F3 cells were continuously positive for zonula occludens-2 (ZO-2), cytokeratin and vimentin, whereas they expressed the TGC specific marker PL only in the first two passages. F3 cell growth was accelerated in medium supplied with epidermal growth factor (EGF). EGF-stimulated proliferation was mediated through activation of Ras and the phosphorylation of mitogen-activated protein kinase (MAPK) 42 and 44. In conclusion, the F3 cell line shows several in vivo characteristics of bovine cotyledonary trophoblast cells. The response to EGF stimulation indicates that EGF plays a role during bovine placentation, and illustrated that F3 cells may provide a valuable tool for further mechanistic studies elucidating the feto-maternal interplay.
在牛胎盘组织中，多核滋养层巨细胞（TGC）由单核滋养层细胞演变而来，对于胎-母相互作用至关重要，因为它们具有内分泌活性，并且能够迁移并与绒毛膜上皮细胞融合。与绒毛膜上皮细胞不同，牛滋养层细胞的分离和培养很复杂，因为它们在长时间培养过程中会停止表达其特定产物，如胎盘催乳素（PL）。在本研究中，我们旨在建立一个针对牛胎盘的体外模型的牛绒毛膜滋养层细胞系，这是我们的长期目标。因此，我们测试了重要信号通路的功能活性。从雄性胎儿的牛胎盘中分离出原代滋养层细胞，并成功地进行了传代和冷冻保存。获得的细胞系命名为 F3，在所有传代中均显示出上皮形态和少量特征性双核巨细胞。通过扩增 Y 染色体特异性 DNA 序列和存在 PL mRNA，验证了 F3 细胞的滋养层来源。免疫荧光表明，F3 细胞持续对紧密连接蛋白-2（ZO-2）、细胞角蛋白和波形蛋白呈阳性，而仅在前两个传代中表达 TGC 特异性标志物 PL。在含有表皮生长因子（EGF）的培养基中，F3 细胞的生长速度加快。EGF 刺激的增殖是通过激活 Ras 和丝裂原活化蛋白激酶（MAPK）42 和 44 的磷酸化来介导的。总之，F3 细胞系显示出牛绒毛膜滋养层细胞的一些体内特征。对 EGF 刺激的反应表明 EGF 在牛胎盘形成过程中发挥作用，并表明 F3 细胞可能为进一步阐明胎-母相互作用的机制研究提供有价值的工具。
