Furuya S, Nagata R, Ozaki Y, Furuya K, Nakayama T, Nagahama M
National Institute for Physiological Sciences, Myodaiji, Okazaki, Japan.
Anat Embryol (Berl). 1997 Feb;195(2):113-26. doi: 10.1007/s004290050030.
A monoclonal antibody was developed, using cultured subepithelial fibroblasts of rat duodenal villi as the antigenic material, by in vitro immunization. Hybridomas were selected on cryosections of rat brain and small intestine using indirect immunofluorescence techniques. The monoclonal antibody, termed 8E1, was very useful to label GFAP-positive astrocytes in the adult rat brain, subepithelial fibroblasts of intestinal villi and Thuneberg's fibroblast-like interstitial cells associated with the myenteric plexus layer. These cells are known to be stellate in shape, forming a cellular network with extended cell processes that communicate via gap junctions. Immature astrocytes such as radial fibers were not immunolabeled with mAb-8E1. Immunoelectron microscopy revealed clustered gold labeling of 8E1 antigen on the intermediate filaments of cultured astrocytes and cultured subepithelial fibroblasts. This staining pattern was different from the continuous and linear gold labeling of GFAP and vimentin. MAb-8E1 did not immunoblot purified human brain GFAP nor bovine lens vimentin, and so was not neutralized by preabsorption with these molecules. Immunoblot analysis after SDS-PAGE showed that the antigen recognized by mAb-8E1 was a Mr 50K protein in the rat cerebra, and a Mr 56K protein in the cultured subepithelial fibroblasts. This study showed that astrocytes and some types of stellate cells share distinct antigenic properties in the components of their intermediate filaments.
利用大鼠十二指肠绒毛培养的上皮下成纤维细胞作为抗原物质，通过体外免疫制备了一种单克隆抗体。使用间接免疫荧光技术在大鼠脑和小肠的冰冻切片上筛选杂交瘤。这种单克隆抗体命名为8E1，对标记成年大鼠脑中的GFAP阳性星形胶质细胞、肠绒毛的上皮下成纤维细胞以及与肌间神经丛层相关的图内伯格成纤维细胞样间质细胞非常有用。已知这些细胞呈星状，通过缝隙连接形成具有延伸细胞突起的细胞网络。诸如放射状纤维等未成熟星形胶质细胞不能被mAb - 8E1免疫标记。免疫电子显微镜显示，8E1抗原在培养的星形胶质细胞和培养的上皮下成纤维细胞的中间丝上呈簇状金标记。这种染色模式与GFAP和波形蛋白的连续线性金标记不同。MAb - 8E1不能对纯化的人脑GFAP和牛晶状体波形蛋白进行免疫印迹，因此也不会被这些分子的预吸附所中和。SDS - PAGE后的免疫印迹分析表明，mAb - 8E1识别的抗原在大鼠脑中是一种分子量为50K的蛋白质，在培养的上皮下成纤维细胞中是一种分子量为56K的蛋白质。这项研究表明，星形胶质细胞和某些类型的星状细胞在其中间丝成分中具有不同的抗原特性。
