Kliman H J, Nestler J E, Sermasi E, Sanger J M, Strauss J F
Endocrinology. 1986 Apr;118(4):1567-82. doi: 10.1210/endo-118-4-1567.
Highly purified functional cytotrophoblasts have been prepared from human term placentae by adding a Percoll gradient centrifugation step to a standard trypsin-DNase dispersion method. The isolated mononuclear trophoblasts averaged 10 microns in diameter, with occasional cells measuring up to 20-30 microns. Viability was greater than 90%. Transmission electron microscopy revealed that the cells had fine structural features typical of trophoblasts. In contrast to syncytial trophoblasts of intact term placentae, these cells did not stain for hCG, human placental lactogen, pregnancy-specific beta 1-glycoprotein or low mol wt cytokeratins by immunoperoxidase methods. Endothelial cells, fibroblasts, or macrophages did not contaminate the purified cytotrophoblasts, as evidenced by the lack of immunoperoxidase staining with antibodies against vimentin or alpha 1-antichymotrypsin. The cells produced progesterone (1 ng/10(6) cells . 4 h), and progesterone synthesis was stimulated up to 8-fold in the presence of 25-hydroxycholesterol (20 micrograms/ml). They also produced estrogens (1360 pg/10(6) cells . 4 h) when supplied with androstenedione (1 ng/ml) as a precursor. When placed in culture, the cytotrophoblasts consistently formed aggregates, which subsequently transformed into syncytia within 24-48 h after plating. Time lapse cinematography revealed that this process occurred by cell fusion. The presumptive syncytial groups were proven to be true syncytia by microinjection of fluorescently labeled alpha-actinin, which diffused completely throughout the syncytial cytoplasm within 30 min. Immunoperoxidase staining of cultured trophoblasts between 3.5 and 72 h after plating revealed a progressive increase in cytoplasmic pregnancy-specific beta 1-glycoprotein, hCG, and human placental lactogen concomitant with increasing numbers of aggregates and syncytia. At all time points examined, occasional single cells positive for these markers were identified. RIA of the spent culture media for hCG revealed a significant increase in secreted hCG, paralleling the increase in hCG-positive cells and syncytia identified by immunoperoxidase methods. We conclude that human cytotrophoblasts differentiate in culture and fuse to form functional syncytiotrophoblasts.
通过在标准的胰蛋白酶 - DNA酶分散方法中加入Percoll梯度离心步骤，已从足月人胎盘中制备出高度纯化的功能性细胞滋养层细胞。分离出的单核滋养层细胞直径平均为10微米，偶尔有细胞直径可达20 - 30微米。活力大于90%。透射电子显微镜显示这些细胞具有典型的滋养层细胞精细结构特征。与完整足月胎盘的合体滋养层细胞不同，通过免疫过氧化物酶方法，这些细胞不表达人绒毛膜促性腺激素（hCG）、人胎盘催乳素、妊娠特异性β1 - 糖蛋白或低分子量细胞角蛋白。免疫过氧化物酶染色显示，用抗波形蛋白或α1 - 抗胰凝乳蛋白酶抗体染色时无阳性反应，证明纯化的细胞滋养层细胞未被内皮细胞、成纤维细胞或巨噬细胞污染。这些细胞可产生孕酮（1纳克/10⁶个细胞·4小时），在25 - 羟胆固醇（20微克/毫升）存在的情况下，孕酮合成可被刺激增加至8倍。当以前体雄烯二酮（1纳克/毫升）提供时，它们还可产生雌激素（1360皮克/10⁶个细胞·4小时）。当置于培养中时，细胞滋养层细胞持续形成聚集体，接种后24 - 48小时内这些聚集体随后转化为合体滋养层。延时摄影显示这个过程是通过细胞融合发生的。通过微注射荧光标记的α - 辅肌动蛋白证明推定的合体滋养层群体是真正的合体滋养层，荧光标记的α - 辅肌动蛋白在30分钟内完全扩散到整个合体滋养层细胞质中。对接种后3.5至72小时的培养滋养层细胞进行免疫过氧化物酶染色显示，随着聚集体和合体滋养层数量的增加，细胞质中妊娠特异性β1 - 糖蛋白、hCG和人胎盘催乳素逐渐增加。在所有检测的时间点，都能识别出偶尔的单个细胞对这些标志物呈阳性。对用过的培养基进行hCG放射免疫分析显示，分泌的hCG显著增加，这与免疫过氧化物酶方法鉴定的hCG阳性细胞和合体滋养层的增加相一致。我们得出结论，人细胞滋养层细胞在培养中分化并融合形成功能性合体滋养层细胞。
