Bethea C L
Endocrinology. 1984 Aug;115(2):443-51. doi: 10.1210/endo-115-2-443.
To determine the direct effect of estrogen on primate PRL production, cultures of dispersed monkey pituitary cells were established in serum-free medium on an extracellular matrix secreted by bovine corneal endothelial cells. Medium PRL levels were monitored by RIA every fourth day after plating. PRL secretion was maintained for 24-28 days in cultures from female monkeys and for 16-20 days in cultures from male monkeys using a 1:1 mixture of Dulbecco's Modified Eagle's Medium H-16 (DME) and Ham's F-12 medium (F12) supplemented with insulin (I), transferrin (T), PTH, T4, fibroblastic growth factor, putrescine, ethanolamine, lipids (oleic, lecithin, and cholesterol), selenium (S), and cadmium (C). The mean percent changes in medium PRL of seven cultures of female pituitaries and two cultures of male pituitaries compared to the first sample (day 4) were: +26% (day 8), +17% (day 12), +11% (day 16), -4% (day 20), -13% (day 24), and -24% (day 28). The elimination of supplements except ITSC did not appear to affect PRL secretion in a culture of cells from ovariectomized female monkeys, but secretion in two cultures of male pituitary cells declined after day 12. Addition of 10(-8) M estrogen to three cultures of male pituitary cells in DME/F12 containing all supplements significantly elevated PRL over control levels and, in addition, prevented the decline observed in control wells of one culture for 28 days. In two cultures of male pituitary cells maintained in DME/F12 plus ITSC, estrogen significantly elevated medium PRL levels for the 28-day period, but did not totally prevent the decline observed in control wells after day 12. In two cultures of female pituitary cells maintained in DME/F12 plus all supplements and in three cultures of female pituitary cells maintained in DME/F12 plus ITSC, medium PRL levels were significantly higher when estrogen was present. However, estrogen addition had no effect on the pattern of PRL secretion in two cultures of female pituitary cells maintained in DME/F12 plus 10% charcoal-treated fetal calf serum. In five of seven cultures, the presence of estrogen for 28 days resulted in a significantly higher cellular content of PRL. These experiments suggest that estrogen can directly increase PRL production by primate mammotrophs and that this effect is best seen in serum-free medium. In summary, extracellular matrix and serum-free medium provide an adequate in vitro environment for studies of PRL processing by primate pituitary cells for periods up to 1 month. In this system, estrogen effectively elevated PRL secretion and cell content.
为了确定雌激素对灵长类动物催乳素（PRL）分泌的直接影响，在由牛角膜内皮细胞分泌的细胞外基质上，于无血清培养基中建立了分散的猴垂体细胞培养体系。接种后每隔四天通过放射免疫分析（RIA）监测培养基中PRL的水平。使用含有胰岛素（I）、转铁蛋白（T）、甲状旁腺激素（PTH）、甲状腺素（T4）、成纤维细胞生长因子、腐胺、乙醇胺、脂质（油酸、卵磷脂和胆固醇）、硒（S）和镉（C）的杜氏改良伊格尔培养基H-16（DME）和哈姆氏F-12培养基（F12）的1:1混合物，雌性猴子的培养物中PRL分泌维持24 - 28天，雄性猴子的培养物中PRL分泌维持16 - 20天。与第一个样本（第4天）相比，七份雌性垂体培养物和两份雄性垂体培养物的培养基中PRL的平均变化百分比为：第8天 +26%，第12天 +17%，第16天 +11%，第20天 -4%，第24天 -13%，第28天 -24%。在去卵巢雌性猴子的细胞培养物中，去除除ITSC之外的补充剂似乎不影响PRL分泌，但两份雄性垂体细胞培养物在第12天后分泌量下降。在含有所有补充剂的DME/F12中，向三份雄性垂体细胞培养物中添加10⁻⁸ M雌激素，显著提高了PRL水平，使其高于对照水平，此外，还防止了一份培养物的对照孔中观察到的28天内的下降。在DME/F12加ITSC中维持的两份雄性垂体细胞培养物中，雌激素在28天期间显著提高了培养基中PRL水平，但没有完全防止第12天后对照孔中观察到的下降。在DME/F12加所有补充剂中维持的两份雌性垂体细胞培养物以及在DME/F12加ITSC中维持的三份雌性垂体细胞培养物中，存在雌激素时培养基中PRL水平显著更高。然而，在DME/F12加10%经活性炭处理的胎牛血清中维持的两份雌性垂体细胞培养物中，添加雌激素对PRL分泌模式没有影响。在七份培养物中的五份中，雌激素存在28天导致PRL的细胞含量显著更高。这些实验表明，雌激素可直接增加灵长类动物促乳素细胞的PRL分泌，并且这种作用在无血清培养基中最为明显。总之，细胞外基质和无血清培养基为长达1个月的灵长类垂体细胞PRL加工研究提供了合适的体外环境。在这个系统中，雌激素有效地提高了PRL分泌和细胞含量。
