Immunocytochemical analysis of prolactin production by monolayer cultures of GH3 rat anterior pituitary tumor cells: I. Long-term effects of stimulation with thyrotropin-releasing hormone (TRH).
Immunocytochemical analysis of prolactin production by monolayer cultures of GH3 rat anterior pituitary tumor cells: I. Long-term effects of stimulation with thyrotropin-releasing hormone (TRH).
复制标题
GH3大鼠垂体前叶肿瘤细胞单层培养物产生催乳素的免疫细胞化学分析：I.促甲状腺激素释放激素（TRH）刺激的长期影响。
DOI：
10.1002/ar.1091970205
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发表时间：
1980
期刊：
影响因子：
--
通讯作者：
TashjianJr,AH
中科院分区：
文献类型：
--
作者：
HoytJr,RF;TashjianJr,AH
促甲状腺激素释放激素 (TRH) 刺激培养的 GH（大鼠垂体前叶肿瘤细胞）中催乳素的产生。为了建立细胞间催乳素分布和细胞内激素浓度 GH 的相关性，将细胞在塑料培养皿中的玻璃盖玻片上生长至平台期密度。通过免疫球蛋白-过氧化物酶桥技术对丙酮固定的带有细胞的盖玻片进行催乳素染色（Mason 等人，'69）；对塑料培养皿上的细胞进行催乳素（微补体固定免疫测定，Tashjian，'73）和蛋白质含量的测定。细胞内催乳素不受丙酮固定和培养基选择的定量影响，通过颗粒状棕色沉淀物进行免疫细胞化学定位，如果用大鼠催乳素预先吸收抗催乳素血清或从方案中省略，则该沉淀物会被消除。在对照和 TRH 处理（10 ng/ml；48 小时）GH 细胞培养物中，用秋水仙碱（5.0 x lo-"；孵育最后 3 小时）使细胞内催乳素最大化。通过光学显微镜将总共 8,500 个细胞分类为催乳素未染色、重度（HI 或中度 (M) 染色）。在对照中，35% 的细胞被染色。 催乳素阳性：6% H 和 2% M。TRH 后，45% 呈阳性：7% H 和 38% M。虽然催乳素阳性细胞分布不均匀，在对照组的各个显微视野中包含 25% 至 469'0 的细胞，但 TRH 增加了所有区域中 M 细胞的比例。 TRH 治疗将催乳素水平提高至对照的 450%，但数学分析将不到 30% 的增加归因于 新的催乳素阳性细胞。我们得出结论，TRH 主要通过提高单个阳性细胞的平均激素含量而不是通过增加致力于产生催乳素的细胞比例来作用于 GH。功能性肿瘤细胞的培养株已在 6ur 实验室进行了十多年的研究。其中之一，OH，是大鼠垂体前叶细胞的克隆，已被证明可用作生产生长激素和 催乳素，事实上，是促甲状腺激素释放激素的生物系统
Thyrotropin-releasing hormone (TRH) stimulates prolactin production in cultured GH, rat anterior pituitary tumor cells. For correlation of cell-by-cell prolactin distribution and intracellular hormone concentration, GH,, cells were grown to plateau-phase density on glass coverslips in plastic dishes. Acetone-fixed, cell-bearing coverslips were stained for prolactin by an immunoglobulin-peroxidase bridge technique (Mason et al.,'69); cells on the plastic dishes were assayed for prolactin (microcomplement fixation immunoassay, Tashjian,'73) and protein content. Intracellular prolactin, unaffected quantitatively by acetone fixation and choice of substratum, was localized immunocytochemically by a granular brown precipitate, abolished if anti-prolactin serum was preabsorbed with rat prolactin or omitted from the protocol. Intracellular prolactin was maximized with colchicine (5.0 x lo-"; final 3 hr of incubation) in control and TRH-treated (10 ng/ml; 48 hr) GH,, cell cultures. A total of 8,500 cells were classified by light microscopy as unstained, heavily (HI or moderately (M) stained for prolactin. In controls, 35% of cells were prolactin-positive: 6% H and 2% M. After TRH, 45% were positive: 7% H and 38% M. Although prolactin-positive cells were unevenly distributed, comprising 25% to 469'0 of cells in individual microscopic fields in controls, TRH increased the proportion of M cells in all areas. TRH treatment raised prolactin levels to 450% of control, but mathematical analysis attributed less than 30% of the increase to new prolactin-positive cells. We conclude that TRH acts on GH, cultures principally by raising the mean hormone content of individual positive cells rather than by increasing the proportion of cells committed to prolactin production.Cultured strains of functional tumor cells have been under study in 6ur laboratory for more than a decade. One of these, the OH,, clone of rat anterior pituitary cells, has proved useful as a model for the production of growth hormone and prolactin and is, in fact, the biological system in which thyrotropin-releasing hor-