Yamaguchi M, Endo H, Thordarson G, Ogren L, Talamantes F
Department of Biology, University of California, Santa Cruz 95064.
Endocrinology. 1992 May;130(5):2897-905. doi: 10.1210/endo.130.5.1315261.
The primary objective of this study was to develop a cell culture system for assessing effects of putative secretagogues on mouse PL-I (mPL-I) secretion. Trophoblast from days 7 to 11 of pregnancy was dispersed in collagenase, and the cells were fractionated on a Percoll gradient and plated on collagen gels in serum-free medium. Cells from days 7-9 of pregnancy yielded five bands on Percoll gradients and those from days 10 and 11 yielded six. mPL-I was present in four of the bands of cells from each day of pregnancy. Cells from day 7 of pregnancy that banded at a density of 1.044 g/ml secreted the largest amount of mPL-I during 5 days of culture. The mPL-I concentration of the medium of these cells increased for the first 3 or 4 days of culture and then declined on the fifth day. mPL-II could not be detected in the medium until the third or fourth day of culture, and its concentration increased thereafter. Cell viability was about 90% at the time of plating, remained at about 80% between days 1 and 4, and then declined on day 5. The cell type that produced mPL-I was identified with the reverse hemolytic plaque assay and by staining with anti-mPL-I antiserum. Both methods indicated that mPL-I was produced by giant cells. The ability of the cells to respond to putative secretagogues was examined using mPL-II and progesterone. mPL-II, at concentrations ranging between 10 ng/ml and 10 micrograms/ml, had no effect on the mPL-I concentration of the medium when it was present for up to 3 days of culture, which suggests that mPL-II does not inhibit mPL-I secretion in vitro. Incubation of the cells in the presence of 100-1000 ng/ml progesterone caused a dose- and time-dependent reduction in the mPL-I concentration of the medium and a decrease in the number of cells that stained with anti-mPL-I antiserum. The effect of progesterone on both endpoints was not apparent until the second day of treatment. These data suggest that progesterone inhibits mPL-I secretion at least in part by inhibiting the differentiation of mPL-I-producing giant cells. The fact that the mPL-I-producing cells responded to progesterone indicates that this culture system will be useful in assessing effects of putative secretagogues on mPL-I secretion.
本研究的主要目的是建立一种细胞培养系统，用于评估假定的促分泌素对小鼠PL-I（mPL-I）分泌的影响。将妊娠第7至11天的滋养层细胞用胶原酶分散，细胞在Percoll梯度上进行分级分离，并接种于无血清培养基中的胶原凝胶上。妊娠第7 - 9天的细胞在Percoll梯度上产生五条带，第10和11天的细胞产生六条带。妊娠各天的细胞中，四条带含有mPL-I。妊娠第7天密度为1.044 g/ml的条带中的细胞在培养5天期间分泌的mPL-I量最大。这些细胞培养基中的mPL-I浓度在培养的前3或4天增加，然后在第5天下降。直到培养的第3或4天，培养基中才检测到mPL-II，此后其浓度增加。接种时细胞活力约为90%，第1至4天保持在约80%，然后在第5天下降。通过反向溶血空斑试验和用抗mPL-I抗血清染色鉴定产生mPL-I的细胞类型。两种方法均表明mPL-I由巨细胞产生。使用mPL-II和孕酮检测细胞对假定促分泌素的反应能力。浓度在10 ng/ml至10 μg/ml之间的mPL-II在培养长达3天存在时，对培养基中mPL-I浓度没有影响，这表明mPL-II在体外不抑制mPL-I分泌。在100 - 1000 ng/ml孕酮存在下孵育细胞导致培养基中mPL-I浓度呈剂量和时间依赖性降低，以及用抗mPL-I抗血清染色的细胞数量减少。孕酮对这两个终点的影响直到处理的第二天才明显。这些数据表明，孕酮至少部分通过抑制产生mPL-I的巨细胞的分化来抑制mPL-I分泌。产生mPL-I的细胞对孕酮有反应这一事实表明，该培养系统将有助于评估假定促分泌素对mPL-I分泌的影响。
