ACTH synthesis and release in pituitary monolayer culture: effect of dexamethasone.
ACTH synthesis and release in pituitary monolayer culture: effect of dexamethasone.
复制标题
垂体单层培养物中促肾上腺皮质激素的合成和释放：地塞米松的作用。
DOI：
10.1152/ajplegacy.1970.219.2.445
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发表时间：
1970
期刊：
影响因子：
--
通讯作者：
W. Rawls
中科院分区：
文献类型：
--
作者：
N. Fleischer;W. Rawls
METHODSPituitaries were removed from male Cheek-Jones rats weighing 100-l 80 g. They were placed in 0.25% trypsin and agitated with a magnetic stirrer for 15 min at 37 C. The dispersed cells were aspirated and placed in media containing 0.35% soybean trypsin inhibitor. Fresh trypsin was added to the undigested pituitary fragments, and the process was twice repeated. The dispersed cells were sedimented by centrifugation, washed 3 times in medium, and resuspended in medium to a concentration of 3-5 X 105/ml. Eagle’s medium supplemented with 10% fetal bovine serum, penicillin 100 U/ml, streptomycin 100 pg/ml, and 0.75 g/liter of sodium bicarbonate were used throughout the study. One-milliliter amounts of the cell suspension were placed in constricted culture tubes which were stoppered and incubated at 37 C. Seventy-two hours later the medium and nonadherent cells were decanted. The cells adherent to the culture tubes were washed with medium, and 1 ml of the medium was readded; medium readded to one-half of the tubes contained dexamethasone-Z-Pod. In some experiments either corticosterone or progesterone was substituted for dexamethasone. The tubes were stoppered and incubated at 37 C. No further medium changes were made. At l-or Z-day intervals, four culture tubes containing cells incubated in the presence of glucocorticoids and four culture tubes containing steroid-free medium were harvested. Cell numbers were determined in duplicate cultures by trypsinizing the monolayers and counting the monodispersed cells in a hemocytometer. Studies of ACTH production and distribution were performed in duplicate cultures in the following manner. The medium from each tube was decanted and saved for estimating the extracellular quantity of ACTH. The cells which were adherent to the culture tubes were washed twice with saline and lysed by adding 1 ml of distilled water followed by vigorous agitation and freezing and thawing. Lysates were utilized for estimating cellular protein by the method of Lowry et al.(9) and for estimating the intracellular level of ACTH.
