Bridger P S, Menge C, Leiser R, Tinneberg H-R, Pfarrer C D
Department of Veterinary Anatomy, Histology and Embryology, Justus-Liebig-University, Frankfurter Strasse 98, D-35392 Giessen, Germany.
Placenta. 2007 Nov-Dec;28(11-12):1110-7. doi: 10.1016/j.placenta.2007.07.002. Epub 2007 Sep 11.
In the bovine synepitheliochorial placenta key sites of fetal-maternal interaction are placentomes consisting of maternal caruncles interdigitating with fetal cotyledons. The aim of this study was to establish an epithelial cell line from caruncles of pregnant cows and to develop a model to study restricted trophoblast invasion, pathogenesis of pregnancy associated diseases and pathways of infection and transport. Primary epithelial cells were isolated, successfully subcultured for 32 passages and cryopreserved at various stages. The cultures were termed bovine caruncular epithelial cell line-1 (BCEC-1). Cytokeratin, zonula occludens-1 protein and vimentin but neither alpha-smooth muscle actin nor desmin were detected by immunofluorescence performed every 5 (+/-1) passages. These results were confirmed by Western blotting. BCEC-1 were then cultured either without matrix or on fibronectin or collagen coated Transwell polyester membrane inserts, respectively, enabling separate access to the basal or apical epithelial compartments. Transmission and scanning electron microscopy of BCEC-1 revealed ultrastructural features also observed in vivo, such as apical microvilli and junctional complexes. Transepithelial electrical resistance (TEER) was measured regularly and revealed an increase with advancing confluence in all cultures. Cultures on coated inserts reached confluence and corresponding TEER-levels at an earlier stage. In addition, the cells were tested negative for bovine virus diarrhoea (BVD) virus, but were permissive for the virus. In conclusion, the BCEC-1 cell line retained characteristics of maternal caruncular epithelial cells as observed in vivo and in primary cell cultures and thus will be a highly useful tool for future studies of pathways of invasion, fetal-maternal communication, transport and infection.
在牛的上皮绒毛膜胎盘,母胎相互作用的关键部位是胎盘小叶,它由与胎儿绒毛叶相互交错的母体肉阜组成。本研究的目的是从怀孕母牛的肉阜中建立一种上皮细胞系,并开发一个模型来研究滋养层细胞的限制性侵袭、妊娠相关疾病的发病机制以及感染和转运途径。分离出原代上皮细胞,成功传代培养32次,并在不同阶段进行冷冻保存。这些培养物被命名为牛肉阜上皮细胞系-1(BCEC-1)。每5(±1)代进行一次免疫荧光检测,结果显示细胞角蛋白、紧密连接蛋白-1和波形蛋白呈阳性,而α-平滑肌肌动蛋白和结蛋白均未检测到。蛋白质免疫印迹法证实了这些结果。然后,分别在无基质的情况下或在纤连蛋白或胶原蛋白包被的Transwell聚酯膜小室上培养BCEC-1,从而能够分别接触上皮细胞的基底侧或顶端侧。BCEC-1的透射电子显微镜和扫描电子显微镜观察显示出在体内也能观察到的超微结构特征,如顶端微绒毛和连接复合体。定期测量跨上皮电阻(TEER),结果显示在所有培养物中,随着汇合程度的增加TEER升高。在包被小室上培养的细胞在较早阶段达到汇合及相应的TEER水平。此外,这些细胞经检测对牛病毒性腹泻(BVD)病毒呈阴性,但对该病毒具有易感性。总之,BCEC-1细胞系保留了在体内和原代细胞培养中观察到的母体肉阜上皮细胞的特征,因此将成为未来研究侵袭途径、母胎通讯、转运和感染的一个非常有用工具。