Irving J A, Lysiak J J, Graham C H, Hearn S, Han V K, Lala P K
Department of Anatomy, University of Western Ontario, London, Canada.
Placenta. 1995 Jul;16(5):413-33. doi: 10.1016/0143-4004(95)90100-0.
We developed a method of propagating pure first trimester human trophoblast cells growing out of primary explants of mechanically derived chorionic villus fragments (Yagel et al, 1989; Graham et al, 1992). We have now extensively characterized these cells during their initial outgrowth and in long-term culture, employing a variety of markers and techniques as outlined below. By double label immunofluorescence using epithelial (cytokeratin) and mesenchymal (vimentin) cell markers, we identified the chorionic villus migrant cell populations as pure trophoblast (39 per cent of outgrowths) or a mixture of trophoblast and fibroblast (61 per cent). Further phenotyping of the pure trophoblast outgrowths by double label immunostaining using anti-cytokeratin antibody and a panel of other primary antisera revealed that these cells exhibit a variety of markers characteristic of extravillous invasive trophoblast cells in situ: insulin-like growth factor (IGF)-II, NDOG-5, proliferating cell nuclear antigen (PCNA), human leucocyte antigen framework antigen (W6/32) and a distinct set of integrins including alpha 1, alpha 3, alpha 5, alpha v and beta 1 subunits and alpha v beta 3/beta 5 vitonectin receptor. They were negative for alpha 6 and beta 4 integrin subunits. Immunogold electron microscopy of explants grown on type IV collagen gel revealed the production of conventional and oncofetal types of fibronectin by mononucleate trophoblast cells and human placental lactogen by multinucleate cells. Immunolabelling, flow cytometry and immunoprecipitation revealed that this phenotypic profile was retained with complete fidelity in the long-term culture; thus, trophoblasts migrating out of first trimester chorionic villus explants and their propagated progeny belong to the invasive extravillous trophoblast of the placenta.
我们开发了一种从机械分离的绒毛膜绒毛片段原代外植体中培养出纯早孕人滋养层细胞的方法（Yagel等人，1989年；Graham等人，1992年）。现在，我们使用如下所述的多种标记物和技术，对这些细胞在最初生长和长期培养过程中进行了广泛的表征。通过使用上皮细胞（细胞角蛋白）和间充质细胞（波形蛋白）标记物的双标记免疫荧光法，我们将绒毛膜绒毛迁移细胞群体鉴定为纯滋养层细胞（占生长物的39%）或滋养层细胞与成纤维细胞的混合物（占61%）。使用抗细胞角蛋白抗体和一组其他一抗血清进行双标记免疫染色，对纯滋养层细胞生长物进行进一步的表型分析，结果显示这些细胞表现出多种原位绒毛外侵袭性滋养层细胞特有的标记物：胰岛素样生长因子（IGF）-II、NDOG-5、增殖细胞核抗原（PCNA）、人白细胞抗原框架抗原（W6/32）以及一组独特的整合素，包括α1、α3、α5、αv和β1亚基以及αvβ3/β5玻连蛋白受体。它们对α6和β4整合素亚基呈阴性。在IV型胶原凝胶上生长的外植体的免疫金电子显微镜检查显示，单核滋养层细胞产生传统型和癌胚型纤连蛋白，多核细胞产生人胎盘催乳素。免疫标记、流式细胞术和免疫沉淀表明，这种表型特征在长期培养中完全保持不变；因此，从早孕绒毛膜绒毛外植体中迁移出来的滋养层细胞及其增殖后代属于胎盘的侵袭性绒毛外滋养层细胞。
