PURIFICATION, CHARACTERIZATION, AND INVITRO DIFFERENTIATION OF CYTOTROPHOBLASTS FROM HUMAN TERM PLACENTAE
PURIFICATION, CHARACTERIZATION, AND INVITRO DIFFERENTIATION OF CYTOTROPHOBLASTS FROM HUMAN TERM PLACENTAE
复制标题
DOI：
10.1210/endo-118-4-1567
复制
发表时间：
1986-04-01
期刊：
影响因子：
4.8
通讯作者：
STRAUSS, JF
中科院分区：
文献类型：
--
作者：
KLIMAN, HJ;NESTLER, JE;STRAUSS, JF
通过在标准的胰酶-DNA酶分散方法中加入Percoll梯度离心法，从人足月胎盘中获得高纯度的功能细胞滋养层细胞。分离的单核滋养层细胞直径平均为10微米，偶尔细胞直径可达20-30微米。存活率大于90%。透射电子显微镜显示细胞具有滋养层细胞的典型结构特征。与完整足月胎盘的合体滋养层细胞相比，这些细胞不表达hCG、人胎盘催乳素、妊娠特异性β1-糖蛋白或低分子细胞角蛋白。内皮细胞、成纤维细胞或巨噬细胞不污染纯化的细胞滋养层细胞，这证明缺乏抗波形蛋白或α1-抗凝乳杆菌胰蛋白酶抗体的免疫过氧化物酶染色。细胞产生孕酮(1 ng/106个细胞)。4小时)，在25-羟基胆固醇(20微克/毫升)存在下，孕酮的合成被刺激到8倍。它们还产生雌激素(1360pg/106细胞.cntdo.当以雄烯二酮(1 ng/ml)为前体时，4h。在培养中，细胞滋养层细胞持续形成聚集体，并在培养后24-48小时内转化为合胞体。时间推移摄影显示这一过程是通过细胞融合发生的。通过显微注射荧光标记的α-肌动蛋白，在30分钟内完全扩散到整个合胞体细胞质，推测的合胞体群被证明是真正的合胞体。培养的滋养层细胞在接种后3.5至72小时的免疫过氧化物酶染色显示，随着聚集体和合胞体数量的增加，胞浆妊娠特异性β1-糖蛋白、hCG和人胎盘催乳素的含量逐渐增加。在检查的所有时间点，偶尔发现单个细胞对这些标志物呈阳性。HCG培养上清液的放射免疫分析显示，hCG的分泌显著增加，与免疫过氧化物酶方法鉴定的hCG阳性细胞和合胞体的增加相平行。我们得出结论，人细胞滋养层细胞在培养过程中分化并融合形成功能性合体滋养层细胞。
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 .mu.m in diameter, with occasional cells measuring up to 20-30 .mu.m. 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/106 cells .cntdot. 4 h), and progesterone synthesis was stimulated up to 8-fold in the presence of 25-hydroxycholesterol (20 .mu.g/ml). They also produced estrogens (1360 pg/106 cells .cntdot. 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 method. We conclude that human cytotrophoblasts differentiate in culture and fuse to form functional syncytiotrophoblasts.