Marelli-Berg F M, Peek E, Lidington E A, Stauss H J, Lechler R I
Department of Immunology, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, W12 ONN, London, UK.
J Immunol Methods. 2000 Oct 20;244(1-2):205-15. doi: 10.1016/s0022-1759(00)00258-1.
The isolation and long-term culture of murine endothelial cells (ECs) has often proven a difficult task. In this paper we describe a quick, efficient protocol for the isolation of microvascular endothelial cells from murine tissues. Murine lung or heart are mechanically minced and enzymatically digested with collagenase and trypsin. The single cell suspension obtained is then incubated with an anti-CD31 antibody, anti-CD105 antibody and with biotinylated isolectin B-4. Pure EC populations are finally obtained by magnetic bead separation using rat anti-mouse Ig- and streptavidin-conjugated microbeads. EC cultures are subsequently expanded and characterised. The surface molecule expression by the primary cultures of murine EC obtained from lung and heart tissue is analysed and compared to that of a murine endothelioma and of primary cultures of murine renal tubular epithelial cells. The phenotype and morphology of these cultures remain stable over 10-15 passages in culture, and no overgrowth of contaminating cells of non-endothelial origin is observed at any stage.
小鼠内皮细胞（ECs）的分离和长期培养常常被证明是一项艰巨的任务。在本文中，我们描述了一种从鼠组织中分离微血管内皮细胞的快速、高效方案。将小鼠肺或心脏进行机械切碎，并用胶原酶和胰蛋白酶进行酶解消化。然后将获得的单细胞悬液与抗CD31抗体、抗CD105抗体以及生物素化的荆豆凝集素B-4一起孵育。最后通过使用大鼠抗小鼠Ig和链霉亲和素偶联的磁珠进行磁珠分离获得纯EC群体。随后对EC培养物进行扩增和鉴定。分析从肺和心脏组织获得的小鼠EC原代培养物的表面分子表达，并将其与小鼠内皮瘤和小鼠肾小管上皮细胞原代培养物的表面分子表达进行比较。这些培养物的表型和形态在培养10 - 15代后保持稳定，并且在任何阶段都未观察到非内皮来源污染细胞的过度生长。
