ENRICHMENT OF CORTICOTROPES BY COUNTERFLOW CENTRIFUGATION
ENRICHMENT OF CORTICOTROPES BY COUNTERFLOW CENTRIFUGATION
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DOI：
10.1210/endo-123-6-2885
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发表时间：
1988-12-01
期刊：
影响因子：
4.8
通讯作者：
UNABIA, G
中科院分区：
文献类型：
--
作者：
CHILDS, GV;LLOYD, JM;UNABIA, G
垂体前叶促肾上腺皮质激素仅占混合垂体细胞群的9-10%。然而，它们的小尺寸阻碍了它们的富集，因为它们不能从更丰富的PRL和GH细胞中分离出来。它们可以通过肾上腺切除术诱导增大，本报告描述了从小垂体细胞亚群中分离出较大的crh反应性促肾上腺皮质细胞。在含有桑德森室的洗脱器中进行逆流离心分离，桑德森室设计用于分离15 .mu以下的小细胞。直径为M。促肾上腺皮质激素最初以低于30 ml/min的流速与小于12.5 .mu的其他细胞一起洗脱。直径为M。然后用0.5 nM CRH刺激2-4小时，并使用更高的流速将增大的促肾上腺皮质激素从未刺激的细胞中分离出来。在五个单独的实验中，整个小细胞池的再营养产生了60%的促肾上腺皮质激素的富集。然而，当池分为小细胞（以20 ml/min洗脱）和中等细胞（以30 ml/min洗脱），并分别洗脱两个池时，在8个单独的实验中，富集量增加到90%以上的促肾上腺皮质激素。这些促肾上腺皮质激素群体在培养中可保持富集达14天。他们也在逆向溶血斑块试验中分泌识别ACTH-(25-39)。ACTH和。beta的双重标签。-内啡肽显示，60%的促肾上腺皮质激素储存两种肽，而30%只储存ACTH， 10%只储存-内啡肽。当比较小型和中型促肾上腺皮质激素时，没有发现储存模式的差异。因此，这些研究首次报道了通过逆流离心产生crh反应性促肾上腺皮质激素的富集部分，并首次报道了ACTH和β -内啡肽的异质储存。富集组分的使用促进了对8000多种促肾上腺皮质激素异质储存模式的分析。
Anterior pituitary corticotropes represent only 9-10% of mixed pituitary cell population. However, their small size precludes their enrichment because they cannot be separated from the more abundant PRL and GH cells. They can be induced to enlarge by adrenalectomy, and this report describes the separation of larger CRH-responsive corticotropes from a subpopulation of small pituitary cells. The separation was done by counterflow centrifugation in an elutriator containing the Sanderson chamber which was designed to separate small cells under 15 .mu.m in diameter. The corticotropes were initially eluted at flow rates under 30 ml/min along with other cells less than 12.5 .mu.m in diameter. They were then stimulated for 2-4 h with 0.5 nM CRH and reeluted with the use of higher flow rates to separate the enlarged corticotropes from the unstimulated cells. Reelutriation of the entire pool of small cells produced an enrichment to 60% corticotropes in five separate experiments. However, when the pool was divided into smaller cells (eluted at 20 ml/min) and medium-sized cells (eluted at 30 ml/min), and the two pools were reeluted separately, the enrichment increased to over 90% corticotropes in eight separate experiments. These corticotrope populations remained enriched for up to 14 days in culture. They also secreted in a reverse hemolytic plaque assay that recognizes ACTH-(25-39). The dual labels for ACTH and .beta.-endorphin showed that 60% of the corticotropes stored both peptides, whereas 30% stored only ACTH, and 10% stored only .beta.-endorphin. No differences in storage patterns were seen when small and medium-sized corticotropes were compared. Thus, these studies present the first report of the production of an enriched fraction of CRH-responsive corticotropes by counterflow centrifugation and the first report of heterogeneous storage of ACTH and .beta.-endorphin. The use of enriched fractions facilitated the analysis of these heterogeneous storage patterns in over 8000 corticotropes.