Lobo J O, Bellino F L
Department of Biological Sciences, State University of New York, Buffalo 14260.
J Clin Endocrinol Metab. 1989 Oct;69(4):868-74. doi: 10.1210/jcem-69-4-868.
The establishment of human term trophoblast cells in culture is dependent on the method of cell preparation, growth medium used, and presence of serum. Using freshly isolated term placental cells, we investigated 1) the effects of two different methods of removal of nontrophoblast cells and two culture media on trophoblast aromatase specific activity, cellular morphology, and hCG secretion over 72 h; and 2) the sensitivity of these hormonal parameters to cAMP added 24 h after the initiation of culture. Under conditions in which aromatase is responsive to cAMP, we studied the effect of removing serum after 24 h in culture with serum. After trypsin digestion of placental villi, isolated trophoblast cells were either treated with ammonium chloride (A) or purified on Percoll density gradients (P) and then grown in monolayer culture with medium 199 plus 10% fetal bovine serum (M) or Dulbecco's Modified Eagle's Medium (DMEM) plus 20% fetal bovine serum (D). Regardless of the method of treatment or growth medium used, aromatase specific activity increased 10- to 15-fold 24 h after plating, continued to increase from 24-48 h, and then decreased (AM) or remained constant (AD, PM, and PD) from 48-72 h. Addition of cAMP significantly increased activity in DMEM-grown cells (PD or AD) at both 48 and 72 h. Aromatase activity in PM cells grown with cAMP increased at 48 h, then decreased to near-control levels by 72 h; however, in AM cells, no response to cAMP was observed relative to control cells. Secretion of hCG was suppressed for the first 48 h in all cultures, but increased by 72 h (greatest increase in AM cultures). cAMP significantly increased hCG secretion 48 h after its addition under all conditions. We further evaluated the response of the Percoll-purified DMEM-grown cells (PD) to cAMP after serum removal at 24 h. Cells deprived of serum showed significantly higher aromatase specific activity over the entire culture period compared with serum-grown cells. Secretion of hCG increased 2- or 3-fold in the presence or absence of serum, respectively, after 72 h in culture. cAMP increased aromatase specific activity by 1.8- and 1.4-fold at 48 h and by 2.5- and 2.4-fold at 72 h in serum-containing and serum-free cells, respectively. The secretion of hCG increased 11- to 14-fold at 72 h under both serum-containing and serum-free conditions, respectively. The results show that cAMP can act as an intracellular messenger in the regulation of both aromatase activity and hCG secretion.(ABSTRACT TRUNCATED AT 400 WORDS)
人足月滋养层细胞在培养中的建立取决于细胞制备方法、所用生长培养基以及血清的存在。我们使用新鲜分离的足月胎盘细胞，研究了：1）两种不同去除非滋养层细胞的方法和两种培养基对滋养层芳香化酶比活性、细胞形态以及72小时内hCG分泌的影响；2）培养开始24小时后添加cAMP对这些激素参数的敏感性。在芳香化酶对cAMP有反应的条件下，我们研究了在含血清培养24小时后去除血清的影响。胎盘绒毛经胰蛋白酶消化后，分离的滋养层细胞要么用氯化铵处理（A），要么在Percoll密度梯度上纯化（P），然后在添加10%胎牛血清的199培养基（M）或添加20%胎牛血清的杜氏改良 Eagle培养基（DMEM）（D）中进行单层培养。无论采用何种处理方法或生长培养基，接种后24小时芳香化酶比活性增加10至15倍，在24至48小时继续增加，然后在48至72小时下降（AM）或保持恒定（AD、PM和PD）。添加cAMP在48小时和72小时均显著增加了在DMEM中培养的细胞（PD或AD）的活性。在添加cAMP的情况下培养的PM细胞中，芳香化酶活性在48小时增加，然后在72小时降至接近对照水平；然而，在AM细胞中，相对于对照细胞未观察到对cAMP的反应。在所有培养物中，hCG分泌在最初48小时受到抑制，但在72小时增加（AM培养物中增加最多）。在所有条件下，添加cAMP后48小时显著增加hCG分泌。我们进一步评估了在24小时去除血清后，Percoll纯化的、在DMEM中培养的细胞（PD）对cAMP的反应。与含血清培养的细胞相比，去除血清的细胞在整个培养期间显示出显著更高的芳香化酶比活性。培养72小时后，无论有无血清，hCG分泌分别增加2倍或3倍。在含血清和无血清的细胞中，cAMP在48小时分别使芳香化酶比活性增加1.8倍和1.4倍，在72小时分别增加2.5倍和2.4倍。在含血清和无血清条件下，72小时时hCG分泌分别增加11至14倍。结果表明，cAMP可作为细胞内信使调节芳香化酶活性和hCG分泌。（摘要截断于400字）
