Douglas G C, King B F
Department of Human Anatomy, School of Medicine, University of California, Davis 95616.
J Cell Sci. 1990 May;96 ( Pt 1):131-41. doi: 10.1242/jcs.96.1.131.
The differentiation of isolated human cytotrophoblast cells has been studied by staining cells with anti-desmoplakin antibodies, to reveal cell boundaries, and with anti-nuclear antibodies, to reveal nuclei. During the first 24 h after plating in Ham's/Waymouth medium, mononucleated cytotrophoblast cells began to spread and aggregate, forming colonies. This was accompanied by progressive changes in the pattern of desmoplakin staining. In single cells, desmoplakin was dispersed throughout the cytoplasm. As cells aggregated, desmoplakin was redistributed and formed linear, punctate arrays at regions of cell-cell contact, consistent with desmosome formation. A pavement-like staining pattern was maintained even at 5 days. Double staining for desmoplakin and nuclei revealed that most cells within colonies were mononucleated. When plated in a growth medium originally formulated for keratinocytes, cytotrophoblast cells aggregated and formed desmosomes normally. However, after 48 h, cell diameters were increased and nuclei changed from being evenly distributed to forming clusters within large cells, consistent with syncytiotrophoblast formation. While cells grown in Ham's/Waymouth medium for 2 days could be induced to differentiate by switching to keratinocyte growth medium, cells cultured for 5 days before switching were resistant to the differentiation-inducing effects of the keratinocyte medium. Desmosome-type junctions within colonies of trophoblast cells were unstable and, even after 5 days in culture, could be disrupted by lowering the extracellular Ca2+ concentration. While syncytiotrophoblast formation in keratinocyte growth medium (which contains epidermal growth factor, insulin and hydrocortisone) was accompanied by a 15- to 20-fold increase in chorionic gonadotropin secretion, syncytiotrophoblast formation occurred to a similar extent in keratinocyte basal medium (which does not contain these factors) but with only a twofold increase in chorionic gonadotropin release. These results support the notion that biochemical and morphological differentiation of trophoblast are independent events.
通过用抗桥粒斑蛋白抗体对细胞进行染色以显示细胞边界，并用抗核抗体对细胞进行染色以显示细胞核，对分离出的人细胞滋养层细胞的分化进行了研究。在接种于哈姆氏/韦茅斯培养基后的最初24小时内，单核细胞滋养层细胞开始铺展并聚集，形成集落。这伴随着桥粒斑蛋白染色模式的逐渐变化。在单个细胞中，桥粒斑蛋白分散于整个细胞质中。随着细胞聚集，桥粒斑蛋白重新分布，并在细胞 - 细胞接触区域形成线性、点状阵列，这与桥粒的形成一致。即使在5天时仍保持铺路石样染色模式。桥粒斑蛋白和细胞核的双重染色显示集落内的大多数细胞为单核。当接种于最初为角质形成细胞配制的生长培养基中时，细胞滋养层细胞正常聚集并形成桥粒。然而，48小时后，细胞直径增大，细胞核从均匀分布变为在大细胞内形成簇，这与合体滋养层细胞的形成一致。虽然在哈姆氏/韦茅斯培养基中培养2天的细胞通过转换到角质形成细胞生长培养基可被诱导分化，但在转换前培养5天的细胞对角质形成细胞培养基的分化诱导作用具有抗性。滋养层细胞集落内的桥粒型连接不稳定，即使在培养5天后，降低细胞外钙离子浓度也可使其破坏。虽然在角质形成细胞生长培养基（含有表皮生长因子、胰岛素和氢化可的松）中合体滋养层细胞的形成伴随着绒毛膜促性腺激素分泌增加15至20倍，但在角质形成细胞基础培养基（不含有这些因子）中也发生了类似程度的合体滋养层细胞形成，但绒毛膜促性腺激素释放仅增加两倍。这些结果支持了滋养层细胞的生化和形态分化是独立事件的观点。
