Salomon D, Meda P
Exp Cell Res. 1986 Feb;162(2):507-20. doi: 10.1016/0014-4827(86)90354-x.
A reverse hemolytic plaque assay was developed to visualize insulin release from individual adult pancreatic B cells. Cells obtained by mechanical dispersion of isolated rat islets of Langerhans were mixed with protein A-coated sheep red blood cells and incubated in the presence of an anti-insulin serum, under conditions known to affect insulin release. The cell mixture was further incubated with complement and finally fixed. Insulin release was revealed by the presence of hemolytic plaques which resulted from the complement-mediated lysis of red blood cells bearing insulin-anti-insulin complexes bound to protein A. Quantitation of hemolytic plaques around trypan blue-unstained and immunohistochemically identified B cells showed that stimulation of insulin release results in the recruitment of increasing numbers of secreting B cells as well as in the enhanced response of individual B cells. Reverse changes occur upon inhibition of insulin release. Comparison of freshly dispersed and one-day-cultured preparations did not reveal significant differences in the secretory response of undamaged B cells. In both preparations, single B cells responded to secretagogues in smaller proportions and to a lesser extent than clusters in which B cells had either maintained or restored contacts and junctional communication with their neighbours. However, the overall preponderant response of clusters was less than expected from the number of individually secreting B cells they contained. The data show that B cells are heterogeneous in terms of their ability to release insulin and provide evidence that cell-to-cell adhesion and/or junctional communication regulate hormone secretion from individual B cells.
开发了一种反向溶血空斑试验来观察成年胰腺β细胞中胰岛素的释放。通过机械分散分离的大鼠胰岛获得的细胞与蛋白A包被的绵羊红细胞混合，并在已知会影响胰岛素释放的条件下，在抗胰岛素血清存在下孵育。细胞混合物进一步与补体孵育，最后固定。胰岛素释放通过溶血空斑的存在得以显示，溶血空斑是由补体介导的带有与蛋白A结合的胰岛素 - 抗胰岛素复合物的红细胞裂解所致。对台盼蓝未染色且经免疫组织化学鉴定的β细胞周围溶血空斑的定量分析表明，胰岛素释放的刺激导致分泌β细胞数量增加以及单个β细胞反应增强。胰岛素释放受抑制时则出现相反变化。新鲜分散的制剂和培养一天的制剂比较显示，未受损β细胞的分泌反应无显著差异。在这两种制剂中，单个β细胞对促分泌剂的反应比例较小且程度较低于β细胞与相邻细胞保持或恢复了接触和连接通讯的细胞簇。然而，细胞簇的总体优势反应低于根据其所含单个分泌β细胞数量预期的值。数据表明β细胞在胰岛素释放能力方面存在异质性，并提供了细胞间粘附和/或连接通讯调节单个β细胞激素分泌的证据。
