Prolactin cell subpopulations separated on discontinuous Percoll gradient: an immunocytochemical, biochemical, and physiological characterization.
Prolactin cell subpopulations separated on discontinuous Percoll gradient: an immunocytochemical, biochemical, and physiological characterization.
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在不连续 Percoll 梯度上分离催乳素细胞亚群：免疫细胞化学、生物化学和生理学特征。
DOI：
10.1210/endo-123-3-1619
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发表时间：
1988
期刊：
影响因子：
4.8
通讯作者：
L. Vanhaelst
中科院分区：
文献类型：
--
作者：
B. Velkeniers;E. Hooghe;R. Hooghe;A. Belayew;G. Smets;A. Claeys;P. Robberecht;L. Vanhaelst
本文报道了一种从大鼠垂体前叶分离PRL细胞的方法。解离后，将细胞在Percoll梯度上离心。回收了三层。使用免疫细胞化学（使用6种垂体激素的抗血清）和原位杂交[使用与PRL或GH信使RNA（mRNA）互补的DNA]评价不同层的组成。两种方法得出的值相同。PRL细胞在较低密度层（层1）中以良好的产率（即初始细胞悬浮液的总PRL细胞的81%）回收，并且另外显著富集（实际上层1中85%的细胞被PRL染色）。第二层（第2层：中等密度）含有大部分剩余的PRL细胞，然而，这些细胞主要被GH细胞和未对任何已知垂体激素染色的细胞严重污染。第三层（第3层：密度较高）富含GH细胞至93%（然而，仅占初始垂体GH细胞的10%）。此外，通过RIA测定培养基和细胞裂解物中的PRL和GH。在[35S]蛋氨酸存在下培养后，通过聚丙烯酰胺凝胶电泳和放射自显影监测激素生物合成。这些实验证实，第1层富集含有和产生PRL的细胞，并耗尽GH细胞。第2层细胞含有和产生更多的GH比PRL。PRL细胞从层1多巴胺和血管活性肠多肽以相同的方式作为PRL细胞在未分离的垂体细胞群。相反，第2层PRL细胞的基础分泌率较低，但对血管活性肠肽的反应较高。除非这代表非PRL细胞的旁分泌效应，否则第2层中的PRL细胞表现出不同的性质，因此可能形成PRL细胞的不同亚群。
A single-step procedure was devised to separate PRL cells from the rat anterior pituitary gland. After dissociation, cells were centrifuged on a Percoll gradient. Three layers were recovered. The composition of the different layers was evaluated using immunocytochemistry (with antisera to the six pituitary hormones), and in situ hybridization [with DNA complementary to PRL or to GH messenger RNA (mRNA)]. Both methods yielded identical values. PRL cells were recovered in the lower density layer (layer 1) with a good yield (that is 81% of the total PRL cells of the initial cell suspension) and in addition, markedly enriched (indeed 85% of the cells in layer 1 stained for PRL). A second layer (layer 2: intermediate density) contained most of the remaining PRL cells which were, however, heavily contaminated mainly by GH cells and cells that did not stain for any of the known pituitary hormones. A third layer (layer 3: higher density) was enriched in GH cells to 93% (representing, however, only 10% of the initial pituitary GH cells). In addition, PRL and GH were measured by RIA in culture medium and in cell lysates. Hormone biosynthesis was monitored by polyacrylamide gel electrophoresis and autoradiography after culture in the presence of [35S]methionine. These experiments confirmed that layer 1 was enriched in cells containing, and producing, PRL and depleted from GH cells. Cells in layer 2 contained and produced more GH than PRL. PRL cells from layer 1 responded to dopamine and to vasoactive intestinal polypeptide in the same way as PRL cells in the unseparated pituitary cell population. In contrast PRL cells in layer 2 had a lower basal secretion rate but a higher response to vasoactive intestinal polypeptide. Unless this represents a paracrine effect of non-PRL cells, PRL cells in layer 2 exhibit different properties and may therefore form a distinct subpopulation of PRL cells.