Rieder H, Ramadori G, Dienes H P, Meyer zum Büschenfelde K H
Department of Internal Medicine, Johannes Gutenberg-Universität Mainz, Federal Republic of Germany.
Hepatology. 1987 Sep-Oct;7(5):856-64. doi: 10.1002/hep.1840070511.
Endothelial liver cells were obtained from guinea pig by enzymatic digestion and centrifugal elutriation. Cells were cultured on gelatin and fibronectin pretreated culture vessels. Endothelial cells were characterized by phase-contrast microscopy, electron microscopy and the presence of Factor VIII-related antigen. Fibronectin secretion was determined in cell-free supernatants by a sensitive and specific ELISA and localized on fixed cultured cells by immunofluorescence. [35S]Methionine endogeneously labeled fibronectin was immunoprecipitated from supernatants and cellular lysates and displayed on sodium dodecyl sulfate polyacrylamide slab gel electrophoresis. After attachment to the culture vessel, one day after plating, endothelial cells start to produce fibronectin as measured by ELISA and demonstrated by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Secretion of fibronectin increases as cells proliferate to form a confluent monolayer. By immunofluorescence, fibronectin is visualized inside permeabilized cells and as a fibrillar network on the cell surface. Underneath the cell bodies, fibronectin-positive material is present as short strands. From supernatants and cellular lysates, fibronectin is immunoprecipitated with an apparent Mr of about 235,000 obviously larger than plasma fibronectin with an Mr of 220,000, which behaves electrophoretically like fibronectin isolated from early hepatocyte cultures. As endothelial cells incorporate [3H]fucose in fibronectin, whereas hepatocytes do not, we conclude that endothelial cells in contrast to hepatocytes produce cellular fibronectin. Endothelial cells, therefore, are probably the cellular source of the fibronectin present in the space of Disse. The significance of this finding with respect to fibrotic liver disease is discussed.
通过酶消化和离心淘析从豚鼠获取肝内皮细胞。细胞接种于经明胶和纤连蛋白预处理的培养器皿上。采用相差显微镜、电子显微镜以及因子VIII相关抗原的存在情况对内皮细胞进行鉴定。通过灵敏且特异的酶联免疫吸附测定法（ELISA）测定无细胞上清液中的纤连蛋白分泌情况，并通过免疫荧光法将其定位在固定的培养细胞上。从上清液和细胞裂解物中免疫沉淀内源性[35S]甲硫氨酸标记的纤连蛋白，并在十二烷基硫酸钠聚丙烯酰胺平板凝胶电泳上展示。接种培养器皿一天后，内皮细胞贴壁后开始产生纤连蛋白，这通过ELISA测定，并经免疫沉淀和十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳证实。随着细胞增殖形成汇合单层，纤连蛋白的分泌增加。通过免疫荧光法，可在通透处理的细胞内观察到纤连蛋白，并且在细胞表面呈现为纤维状网络。在细胞体下方，纤连蛋白阳性物质以短链形式存在。从上清液和细胞裂解物中免疫沉淀得到的纤连蛋白，其表观分子量约为235,000，明显大于血浆纤连蛋白的分子量220,000，其电泳行为类似于从早期肝细胞培养物中分离的纤连蛋白。由于内皮细胞在纤连蛋白中掺入[3H]岩藻糖，而肝细胞则不掺入，我们得出结论，与肝细胞相比，内皮细胞产生细胞纤连蛋白。因此，内皮细胞可能是狄氏间隙中存在的纤连蛋白的细胞来源。本文讨论了这一发现对于肝纤维化疾病的意义。
