Wynick D, Critchley R, Venetikou M S, Burrin J M, Bloom S R
Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London.
J Endocrinol. 1990 Aug;126(2):269-74. doi: 10.1677/joe.0.1260269.
As the secretory granules of anterior pituitary cells fuse with the cell surface, there would appear to be sufficient hormone present on the cell surface to be labelled by polyclonal hormone antibodies and thus analysed by flow cytometry. We have therefore applied fluorescence-activated cell sorting to these labelled pituitary cells. Percentage purity and depletion of other cell types was assessed by immunocytochemistry and the reverse haemolytic plaque assay (RHPA). Results demonstrate that fluorescence-activated cell sorting allows almost complete purification of functional lactotrophs and somatotrophs to 96.7 +/- 1.7 (S.E.M.)% and 98 +/- 1.0% respectively by immunocytochemistry, and to 95.8 +/- 1.1% and 97 +/- 0.8% respectively by RHPA. Depletion of other anterior pituitary cell types to less than 2% was demonstrated by both immunocytochemistry and RHPA. Fluorescence-activated cell sorting to this degree of purity was routinely possible with cell yields of 91 +/- 3.4%. To obtain such purity/depletion, it was necessary to use specific antisera of high titre, at concentrations which ensured maximal cell-surface labelling associated with maximal stimulation of hormonal secretion by the appropriate hypothalamic stimulatory factor. Separating cells on the basis of the intensity of prolactin cell-surface labelling demonstrated a low level of binding of the prolactin antibody to gonadotrophs (but not of sufficient fluorescence intensity to be sorted into the prolactin enriched population), raising the possibility of prolactin receptors on gonadotrophs. We were unable to demonstrate the presence of mammosomatotrophs in the normal female rat, since purified lactotrophs did not contain or secrete GH nor did purified somatotrophs contain or secrete prolactin.
随着垂体前叶细胞的分泌颗粒与细胞表面融合，细胞表面似乎存在足够的激素，可被多克隆激素抗体标记，从而通过流式细胞术进行分析。因此，我们将荧光激活细胞分选技术应用于这些标记的垂体细胞。通过免疫细胞化学和反向溶血空斑试验（RHPA）评估其他细胞类型的纯度百分比和去除情况。结果表明，荧光激活细胞分选技术通过免疫细胞化学可分别将功能性催乳素细胞和生长激素细胞几乎完全纯化至96.7±1.7（标准误）%和98±1.0%，通过RHPA分别纯化至95.8±1.1%和97±0.8%。免疫细胞化学和RHPA均显示其他垂体前叶细胞类型的去除率低于2%。以91±3.4%的细胞产量常规实现荧光激活细胞分选至如此高的纯度。为了获得这样的纯度/去除率，有必要使用高滴度的特异性抗血清，其浓度要确保最大程度的细胞表面标记，同时与适当的下丘脑刺激因子对激素分泌的最大刺激相关联。根据催乳素细胞表面标记强度分离细胞表明，催乳素抗体与促性腺激素细胞的结合水平较低（但荧光强度不足以分选到富含催乳素的群体中），这增加了促性腺激素细胞上存在催乳素受体的可能性。我们无法在正常雌性大鼠中证明存在乳腺生长激素细胞，因为纯化的催乳素细胞不含有或分泌生长激素，纯化的生长激素细胞也不含有或分泌催乳素。
