Baelde H J, Bergijk E C, Bruijn J A
Department of Pathology, University of Leiden, The Netherlands.
J Clin Lab Immunol. 1990 Sep;33(1):17-20.
Glomerular basement membrane (GBM) antigens used for immunological studies have until now been isolated from human and rat glomeruli but not from mice. However, given the growing awareness of extracellular matrix species-specificity, the need for a purification method of mouse GBM exists. We now report the purification of GBM from isolated mouse glomeruli. 10 ml 0.01 M phosphate buffered saline (PBS) containing 1.25% Fe3O4 was perfused through the aortae of (C57BL10 x DBA/2)F1 mice over 1 min, kidneys were decapsulated and passed through a 0.075 mm mesh metal screen and collected. After pelletting and washing, the tube was placed against one pole of a magnet and the pelleted glomeruli were washed. This procedure was repeated three times. The glomerular suspension was examined by light microscopy, sonicated, and lyophilized. In suspension no free fragments of tubuli or Bowman's capsule were present as observed by light microscopy. Mouse GBM was prepared from the isolated glomeruli using a modification of earlier described methods for human and rat GBM by enzymatic digestion. Elisa studies showed the presence of laminin, type IV collagen, and fibronectin. Monoclonal antibody directed against tubular epithelial antigen gp160 did not react to either mouse or rat GBM. Sera and glomerular eluates from (C57BL10 x DBA/2)F1 mice suffering from chronic graft-versus-host autoimmune disease gave a higher signal against mouse GBM than against rat GBM. Normal rabbit serum, normal mouse serum and normal mouse eluate did not show significant activity against mouse GBM. Immunoblotting showed the presence of both laminin B1 and B2 chains as well as type IV collagen alpha 1 and alpha 2 subunits. In summary, we describe a method by which it is possible to extract mouse GBM with a high purity.
迄今为止，用于免疫学研究的肾小球基底膜（GBM）抗原是从人和大鼠的肾小球中分离出来的，而非从小鼠中获取。然而，鉴于对细胞外基质物种特异性的认识不断提高，需要一种纯化小鼠GBM的方法。我们现在报告从分离的小鼠肾小球中纯化GBM的方法。将含有1.25% Fe3O4的10 ml 0.01 M磷酸盐缓冲盐水（PBS）在1分钟内灌注到（C57BL10×DBA/2）F1小鼠的主动脉中，摘除肾脏包膜，使其通过0.075 mm筛孔的金属筛网并收集。离心沉淀并洗涤后，将试管置于磁体的一极，对沉淀的肾小球进行洗涤。此步骤重复三次。通过光学显微镜检查肾小球悬浮液，进行超声处理并冻干。光学显微镜观察显示，悬浮液中不存在肾小管或鲍曼囊的游离碎片。采用对先前描述的人及大鼠GBM方法进行改进后的酶消化法，从分离的肾小球中制备小鼠GBM。酶联免疫吸附测定（ELISA）研究显示存在层粘连蛋白、IV型胶原和纤连蛋白。针对肾小管上皮抗原gp160的单克隆抗体对小鼠或大鼠GBM均无反应。患有慢性移植物抗宿主自身免疫性疾病的（C57BL10×DBA/2）F1小鼠的血清和肾小球洗脱液对小鼠GBM的信号比对大鼠GBM的信号更强。正常兔血清、正常小鼠血清和正常小鼠洗脱液对小鼠GBM均未显示出显著活性。免疫印迹显示存在层粘连蛋白B1和B2链以及IV型胶原α1和α2亚基。总之，我们描述了一种能够以高纯度提取小鼠GBM的方法。
