Babalola G O, Coutifaris C, Soto E A, Kliman H J, Shuman H, Strauss J F
Department of Obstetrics and Gynecology, University of Pennsylvania, School of Medicine, Philadelphia 19104.
Dev Biol. 1990 Jan;137(1):100-8. doi: 10.1016/0012-1606(90)90011-7.
The syncytial trophoblast of the human placenta forms by the fusion of mononuclear cytotrophoblast cells. Cytotrophoblast cells only fuse with other trophoblastic cells, indicating a specificity to this interaction. To explore the cellular aggregation which precedes fusion, we examined the association of cytotrophoblast cells isolated from term placentae and JEG-3 choriocarcinoma cells, a cytotrophoblast-like cell line, in suspension culture. Cytotrophoblast cells were isolated by dispersion of chorionic villi in trypsin-DNase in Ca2+/Mg2(+)-free medium. JEG-3 cells were released from culture flasks by trypsinization in Versene-EDTA buffer. In suspension culture, each cell type aggregated forming tissue-like masses over a 24-hr period. Transmission electron microscope analysis demonstrated the formation of numerous desmosomes between the aggregated cells. In outgrowth culture, the aggregates created in suspension were maintained as microvilli-covered multicellular structures with hollow cores. The extent of aggregation was dependent upon the concentration of cells in the incubations with greater aggregation occurring with higher cell densities. Aggregation of both cytotrophoblast cells and JEG-3 cells progressed rapidly during the initial 10 hr of incubation and then continued at a slower rate. Aggregation took place in serum-containing and serum-free medium, but was impeded in Ca2+/Mg2(+)-free medium. Incubation of JEG-3 and cytotrophoblast cells in the presence of the protein synthesis inhibitor, cycloheximide, prevented aggregation, whereas the inhibitor of N-linked glycosylation, tunicamycin, did not. The inhibitor of RNA synthesis, actinomycin D, had no effect on the aggregation of the cells during the initial 6 hr of aggregation. These findings suggest that trypsin treatment in Ca2+/Mg2(+)-poor medium removed a protein(s) from the trophoblast cell surface which must be resynthesized for cell-cell association to take place.
人胎盘的合体滋养层细胞由单核细胞滋养层细胞融合形成。细胞滋养层细胞仅与其他滋养层细胞融合，表明这种相互作用具有特异性。为了探究融合前的细胞聚集情况，我们在悬浮培养中检测了从足月胎盘分离的细胞滋养层细胞与JEG-3绒毛膜癌细胞（一种细胞滋养层样细胞系）之间的结合。细胞滋养层细胞通过在无Ca2+/Mg2+的培养基中用胰蛋白酶-脱氧核糖核酸酶分散绒毛膜绒毛来分离。JEG-3细胞通过在Versene-EDTA缓冲液中用胰蛋白酶消化从培养瓶中释放出来。在悬浮培养中，每种细胞类型在24小时内聚集形成组织样团块。透射电子显微镜分析表明，聚集细胞之间形成了许多桥粒。在生长培养中，悬浮培养中形成的聚集体保持为覆盖有微绒毛的中空多核结构。聚集程度取决于培养物中的细胞浓度，细胞密度越高，聚集程度越高。细胞滋养层细胞和JEG-3细胞的聚集在孵育的最初10小时内迅速进行，然后以较慢的速度继续。聚集在含血清和无血清培养基中均可发生，但在无Ca2+/Mg2+的培养基中受到阻碍。在蛋白质合成抑制剂环己酰亚胺存在的情况下孵育JEG-3细胞和细胞滋养层细胞可阻止聚集，而N-连接糖基化抑制剂衣霉素则无此作用。RNA合成抑制剂放线菌素D在聚集的最初6小时内对细胞聚集没有影响。这些发现表明，在低Ca2+/Mg2+培养基中进行胰蛋白酶处理会从滋养层细胞表面去除一种蛋白质，这种蛋白质必须重新合成才能发生细胞间结合。
