Srivastava R K, Gu Y, Zilberstein M, Ou J S, Mayo K E, Chou J Y, Gibori G
Department of Physiology and Biophysics, University of Illinois at Chicago, Illinois 60612-7342, USA.
Endocrinology. 1995 May;136(5):1913-9. doi: 10.1210/endo.136.5.7720638.
The rat decidual tissue is formed by two cell populations, which express different genes and play diverse roles in pregnancy. Cells that decidualize in the antimesometrial region secrete several hormones and serve as a true endocrine gland. Isolation and maintenance of these decidual cells in primary culture is difficult. The goal of these experiments was to develop a cell line to serve as a model to study the expression and regulation of various genes specific to the antimesometrial decidual cells. Decidualization was induced in pseudopregnant rats. The antimesometrial decidua was dissected out, and cells were enzymatically dissociated and were cultured for 18 h at 37 C in RPMI-1640 medium containing 10% fetal bovine serum. Cells were washed repeatedly and then infected with a temperature-sensitive mutant of the simian virus. Transformed cells were maintained at the permissive temperature (33 C) until colonies were identified and harvested. Whereas primary cells in culture did not divide, the cloned decidual cell lines demonstrated transformed features; they multiplied at 33 C and formed multilayers. At the nonpermissive temperature (39 C), cell replication decreased, and after 4 days of culture the cells lost their transformed phenotype and continued to grow as a monolayer similar to primary cells. Cells under these conditions also assumed morphological characteristics similar to antimesometrial cells: polynucleated, large, and having cytoplasm filled with lipid droplets. Interestingly, cells cultured at 39 C that were shifted back to 33 C resumed rapid growth. To determine whether these cells also express messenger RNAs (mRNAs) found in normal antimesometrial decidual cells, the presence of activin beta A mRNA was investigated by Northern analysis and reverse transcription polymerase chain reaction. A single 6.8-kilobase activin beta A transcript was expressed abundantly at both 33 and 39 C, indicating that even when cells are rapidly dividing they express activin beta A. Activin beta B mRNA was also expressed in these cells, although in lower abundance, as were two binding proteins for activin, activin receptor II and follistatin. The activin beta A and beta B genes were responsive to cAMP stimulation in these cells. Since the hallmark of the antimesometrial decidual cells is the secretion of PRL-related hormones, the expression of decidual PRL-related protein and PRL-like protein B was examined. Northern analysis revealed a major 1.2-kilobase transcript of PRL-like protein B expressed equally at both temperatures.(ABSTRACT TRUNCATED AT 250 WORDS)
大鼠蜕膜组织由两种细胞群体构成，它们表达不同基因并在妊娠中发挥不同作用。在子宫系膜对侧区域蜕膜化的细胞分泌多种激素，起着真正内分泌腺的作用。在原代培养中分离并维持这些蜕膜细胞很困难。这些实验的目的是建立一种细胞系，用作研究子宫系膜对侧蜕膜细胞特异的各种基因表达和调控的模型。在假孕大鼠中诱导蜕膜化。解剖出子宫系膜对侧的蜕膜，将细胞酶解后，在含10%胎牛血清的RPMI-1640培养基中于37℃培养18小时。细胞反复洗涤后，用猿猴病毒的温度敏感突变体感染。将转化细胞维持在允许温度（33℃），直到鉴定并收获克隆。培养中的原代细胞不分裂，而克隆的蜕膜细胞系表现出转化特征；它们在33℃增殖并形成多层。在非允许温度（39℃）下，细胞复制减少，培养4天后细胞失去转化表型，继续像原代细胞一样单层生长。在这些条件下的细胞还呈现出与子宫系膜对侧细胞相似的形态特征：多核、体积大，细胞质充满脂滴。有趣的是，在39℃培养后又转移回33℃的细胞恢复快速生长。为了确定这些细胞是否也表达正常子宫系膜对侧蜕膜细胞中发现的信使核糖核酸（mRNA），通过Northern印迹分析和逆转录聚合酶链反应研究激活素βA mRNA的存在情况。在33℃和39℃时均大量表达单一的6.8千碱基激活素βA转录本，表明即使细胞快速分裂时它们也表达激活素βA。激活素βB mRNA在这些细胞中也有表达，不过丰度较低，激活素的两种结合蛋白，激活素受体II和卵泡抑素也是如此。激活素βA和βB基因在这些细胞中对cAMP刺激有反应。由于子宫系膜对侧蜕膜细胞的标志是分泌PRL相关激素，检测了蜕膜PRL相关蛋白和PRL样蛋白B的表达。Northern印迹分析显示，PRL样蛋白B的一个主要1.2千碱基转录本在两个温度下表达水平相同。（摘要截短至250词）
