Vuillez P, René F, Plante M, Hindelang C, Klein M J, Félix J M, Stoeckel M E
Laboratoire de Physiologie, URA CNRS no 1446, Strasbourg, France.
Cell Tissue Res. 1992 Jan;267(1):169-83. doi: 10.1007/BF00318702.
Organotypic cultures, in defined medium, of pituitary primordia obtained from 15-day-old rat fetuses were performed in order to study the in vitro differentiation of melanotrophic cells. The morphological and ultrastructural features of the transplants resembled those of the gland developing in vivo. In situ hybridization on semi-thin sections, using a 35S-labelled oligonucleotide probe, revealed pro-opiomelanocortin-mRNA-containing cells on the first day of culture in the anterior lobe and after 2-3 days in the intermediate lobe. Immunoperoxidase labelling of adjacent sections showed that the same cells reacted with antibodies against alpha-melanocyte-stimulating hormone (alpha MSH), gamma 3MSH and adrenocorticotropic hormone in both lobes. The pro-opiomelanocortin-mRNA-containing cells formed progressively conspicuous areas in the intermediate lobe, which was almost uniformly labelled after 6 days. In the anterior lobe, these cells remained scattered in small cell groups, and colloidal gold immunolabelling showed the progressive disappearance of alpha MSH labelling from the secretory vesicles in cells exhibiting morphological features of adult corticotrophic cells. Both the alpha MSH content of the explants and alpha MSH release into the culture medium increased with time. Treatment with the dopamine agonist bromocriptine induced a strong dose-dependent decrease in alpha MSH secretion, which was significant after 3 days in culture, indicating that dopamine D2 receptors are able to regulate hormonal release of melanotrophic cells at early stages. This system constitutes a suitable model for further studies of factors controlling cell differentiation and cellular interactions involved in histogenesis.
为了研究促黑素细胞的体外分化，我们采用限定培养基对取自15日龄大鼠胎儿的垂体原基进行了器官型培养。移植组织的形态和超微结构特征与体内发育的腺体相似。使用35S标记的寡核苷酸探针在半薄切片上进行原位杂交，结果显示在培养的第一天，前叶就出现了含阿黑皮素原mRNA的细胞，中间叶则在培养2 - 3天后出现。对相邻切片进行免疫过氧化物酶标记显示，两叶中相同的细胞与抗α - 黑素细胞刺激素（α MSH）、γ3MSH和促肾上腺皮质激素的抗体发生反应。含阿黑皮素原mRNA的细胞在中间叶逐渐形成明显的区域，6天后几乎被均匀标记。在前叶，这些细胞仍散在分布于小细胞群中，胶体金免疫标记显示，在呈现成年促肾上腺皮质细胞形态特征的细胞中，α MSH标记从分泌小泡中逐渐消失。外植体的α MSH含量以及释放到培养基中的α MSH均随时间增加。用多巴胺激动剂溴隐亭处理导致α MSH分泌呈强烈的剂量依赖性下降，培养3天后差异显著，这表明多巴胺D2受体能够在早期阶段调节促黑素细胞的激素释放。该系统构成了一个合适的模型，可用于进一步研究控制细胞分化的因素以及组织发生过程中涉及的细胞间相互作用。
