INITIAL ADHESION OF HUMAN-FIBROBLASTS IN SERUM-FREE MEDIUM - POSSIBLE ROLE OF SECRETED FIBRONECTIN
INITIAL ADHESION OF HUMAN-FIBROBLASTS IN SERUM-FREE MEDIUM - POSSIBLE ROLE OF SECRETED FIBRONECTIN
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DOI：
10.1016/0092-8674(79)90300-3
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发表时间：
1979-01-01
期刊：
影响因子：
64.5
通讯作者：
FELD, MK
中科院分区：
文献类型：
--
作者：
GRINNELL, F;FELD, MK
人成纤维细胞在无血清培养基中的初始附着和铺展发生于分泌并吸附在基质表面的细胞纤连蛋白。人皮肤成纤维细胞贴附和传播的组织培养基质中的无血清培养基在60分钟。当潜在的蛋白质吸附位点的基质上覆盖牛血清白蛋白之前，最初的人成纤维细胞附着，其随后的附着到基质被阻止。当底层吸附网站被覆盖后立即初始附着，随后的细胞扩散被阻止。采用单特异性抗冷不溶性球蛋白抗血清，通过间接免疫荧光法研究了纤维连接蛋白在人成纤维细胞表面初始附着和铺展过程中的分布。纤连蛋白的初始外观（10分钟）是在整个细胞表面上的斑点。随着人成纤维细胞的铺展，随机分布的位点消失，随后在细胞基质下表面的斑点中观察到大多数纤连蛋白（60 min）。随后（2-8 h）出现纤维连接蛋白的纤维状图案。在用0.1 M NaOH处理附着的人成纤维细胞后，细胞下方的位点被可视化为基质上的足迹。基质上分泌的纤连蛋白的第二种荧光模式的特征是细胞周围有弥漫性光环，基质上其他地方有非常微弱的弥漫性染色。使用另一种细胞类型（幼仓鼠肾[BHK]细胞）测定是否存在由人成纤维细胞分泌到基质上的因子。人成纤维细胞分泌的BHK细胞的粘附因子的基质上的时间和温度依赖性的方式，和免疫学研究表明，这与冷不溶性球蛋白，血浆形式的纤连蛋白的交叉反应。由人成纤维细胞分泌的调节因子是人成纤维细胞的附着和铺展因子。基质吸附的冷不溶性球蛋白也是人成纤维细胞的附着和铺展因子。基于调节因子在基质上出现的时间和免疫荧光模式，基质上弥散组织的纤连蛋白可能构成细胞附着的位点。在细胞下方出现的纤维连接蛋白的亮点可能代表细胞铺展过程中纤维连接蛋白的重组。
The hypothesis that the initial attachment and spreading of human fibroblasts in serum-free medium occurs to cell fibronectin which was secreted and adsorbed on the substratum surface was tested. Human skin fibroblasts attached and spread on tissue culture substrata in serum-free medium in 60 min. When potential protein adsorption sites on the substratum were covered with bovine serum albumin before initial human fibroblast attachment, their subsequent attachment to the substratum was prevented. When substratum adsorption sites were covered immediately after initial attachment, subsequent cell spreading was prevented. The distribution of fibronectin on human fibroblast surfaces during initial attachment and spreading was studied by indirect immunofluorescence using a monospecific anti-cold-insoluble globulin antiserum. The initial appearance (10 min) of fibronectin was in spots over the entire cell surface. Concomitant with human fibroblast spreading, the random distribution of sites disappeared and most fibronectin was subsequently observed in spots at the cell substratum inferface (60 min). A fibrillar pattern of fibronectin appeared later (2-8 h). The sites beneath the cells were visualized as footprints on the substratum following treatment of the attached human fibroblasts with 0.1 M NaOH. A 2nd fluorescence pattern of fibronectin secreted on the substratum was characterized by a diffuse halo around the cells and a very faint, diffuse staining elsewhere on the substratum. Another cell type (baby hamster kidney [BHK] cells) was used to assay for the presence or absence of the factor secreted by human fibroblasts onto the substratum. Human fibroblasts secreted an adhesion factor for BHK cells onto the substratum in a time- and temperature-dependent fashion, and immunological studies indicated that this cross-reacted with cold-insoluble globulin, the plasma form of fibronectin. The conditioning factor secreted by the human fibroblasts was an attachment and spreading factor for human fibroblasts. Substratum-adsorbed cold-insoluble globulin was also an attachment and spreading factor for human fibroblasts. Based upon the timing of appearance of conditioning factors on the substratum and the immunofluorescence patterns, the diffusely organized fibronectin on the substratum probably constitutes the sites to which cells attach. The bright spots of fibronectin that appear beneath the cells may represent fibronectin reorganization during cell spreading.