Childs G V, Lloyd J, Unabia G, Rougeau D
Department of Anatomy and Neurosciences, University of Texas Medical Branch, Galveston 77551.
Endocrinology. 1989 Nov;125(5):2540-9. doi: 10.1210/endo-125-5-2540.
The purpose of this study was to learn whether enriched populations of corticotropes could be grown without the other pituitary cell types. Corticotropes populations were enriched to 80-90% by counterflow centrifugation in an elutriator with the Sanderson chamber. After initial separation into small, medium, and large fractions, the cells were stimulated for 3 h with 0.5 nM corticotropin-releasing hormone (CRH) and then re-eluted to remove the enlarged corticotropes. More ACTH (6- to 10-fold) was released in media with 10% fetal bovine serum (FBS) than was released in media with no serum. The effects of FBS could not be mimicked by 0.3% BSA. Corticotropes grew in serum-containing media as long as they were plated at a density of at least 2500 cells per well. The corticotropes expanded in size and assumed two major morphological subtypes. Both stored ACTH and beta-endorphin. One subtype was flattened and pleomorphic. The other subtype was stellate with multiple processes. Cell counts showed a 2.5- to 3.8-fold increase in the number of labeled corticotropes during the first 21 days of culture. Then the numbers of cells declined rapidly. Basal secretion of ACTH rose 1.6-fold during the first week, plateaued after 14 days and then declined to less than 30% of first week levels. CRH stimulation produced dose-dependent increases in media ACTH. In 7 day cultures, both basal and stimulated levels of ACTH were similar to those in 7 day cultures of mixed pituitary cells (containing equivalent numbers of corticotropes). Stimulatory effects of CRH were evident for up to 42 days of culture. Arginine vasopressin enhanced CRH-mediated secretion in most cultures in the first week. Pretreatment with glucocorticoids (100 nM corticosterone) for 15 h blocked CRH-mediated secretion in all cultures. The studies showed that corticotropes do not need the other pituitary cell types for basic plating and basal and CRH-mediated secretory responses. Further tests of specific growth factors are needed to learn whether they will maintain function for longer periods.
本研究的目的是了解促肾上腺皮质激素细胞富集群体能否在没有其他垂体细胞类型的情况下生长。在带有桑德森腔室的淘洗器中通过逆流离心将促肾上腺皮质激素细胞群体富集至80 - 90%。在初步分离为小、中、大组分后，用0.5 nM促肾上腺皮质激素释放激素（CRH）刺激细胞3小时，然后再次淘洗以去除增大的促肾上腺皮质激素细胞。与无血清培养基相比，在含有10%胎牛血清（FBS）的培养基中释放的促肾上腺皮质激素（ACTH）更多（6至10倍）。0.3%牛血清白蛋白（BSA）无法模拟FBS的作用。只要以每孔至少2500个细胞的密度接种，促肾上腺皮质激素细胞就能在含血清的培养基中生长。促肾上腺皮质激素细胞体积增大，并呈现出两种主要的形态亚型。两种亚型都储存ACTH和β-内啡肽。一种亚型扁平且形态多样。另一种亚型呈星状，有多个突起。细胞计数显示，在培养的前21天，标记的促肾上腺皮质激素细胞数量增加了2.5至3.8倍。然后细胞数量迅速下降。ACTH的基础分泌在第一周上升了1.6倍，14天后达到平稳，然后下降至第一周水平的不到30%。CRH刺激使培养基中的ACTH呈剂量依赖性增加。在7天的培养中，ACTH的基础水平和刺激水平与混合垂体细胞7天培养（含有等量促肾上腺皮质激素细胞）中的水平相似。CRH的刺激作用在长达42天的培养中都很明显。在第一周，精氨酸加压素在大多数培养物中增强了CRH介导的分泌。用糖皮质激素（100 nM皮质酮）预处理15小时可阻断所有培养物中CRH介导的分泌。研究表明，促肾上腺皮质激素细胞在基础接种以及基础和CRH介导的分泌反应方面不需要其他垂体细胞类型。需要对特定生长因子进行进一步测试，以了解它们是否能在更长时间内维持功能。
