Culp L A, Buniel J F
J Cell Physiol. 1976 May;88(1):89-106. doi: 10.1002/jcp.1040880111.
The effects of serum and coatings of substrate-attached material (SAM, which remains tightly adherent to the substrate after EGTA-mediated removal of cells) on the kinetics of attachment of DNA-radiolabeled BALB/c 3T3. SV40-transformed 3T3, and concanavalin A-selected revertant cells to glass coverlips were studied. The presence of serum in the medium of attaching cells had a marked effect on (1) the initial time lag before stable attachment of cells, (2) the maximum level of attached cells, (3) the stability of attachment, and (4) pseudopodial spread of the cell over the substrate. These serum effects could be mimicked by measuring attachment in medium without serum and with use of serum-preadsorbed or 3T3 SAM-coated coverslips. Enzymatic treatment of serumpreadsorbed substrates indicated that the factor(s) in serum which affects attachment is very trypsin-sensitive. Serum preadsorption of substrates stimulated attachment of SVT2 cells in medium with serum in a manner very similar to the effects of 3T3 SAM coating, while attachment of 3T3 SAM coating, while attachment of 3T3 or revertant cells was unaffected. Slab gel electrophoretic analysis (PAGE-SDS gels) identified eight major serum proteins by Coomassie blue staining (a) which bind to the substrate in the absence of cells and (b) which persist on the substrate after growth to confluence of 3T3 or SVT2 cells; this suggests that major breakdown or serum-adsorbed components does not occur during growth of normal or transformed cells. Seven radioactive SAM proteins were detected by autoradiography in 3T3 or SVT2 SAM electropherograms -- two of which are high molecular weight components which correspond to the glucosamine-radiolabeled hyaluronate proteoglycans observed previously; the remaining five are newly-identified proteins in SAM (one of these proteins appears to be actin). 3T3 and SVT2 cells have unique proportions of these seven components. The data are consistent with the idea that normal or virus-transformed cells do not attach directly to the culture substrate, but to specific classes of substrate-adsorbed serum proteins via deposition of specific classes of cell surface proteins and polysaccharides.
研究了血清以及底物附着材料（SAM，在经乙二醇双乙胺四乙酸（EGTA）介导去除细胞后仍紧密附着于底物）的包被对DNA放射性标记的BALB/c 3T3、SV40转化的3T3以及伴刀豆球蛋白A选择的回复细胞附着于玻璃盖玻片动力学的影响。在贴壁细胞培养基中血清的存在对以下方面有显著影响：（1）细胞稳定附着前的初始时间延迟；（2）附着细胞的最大水平；（3）附着的稳定性；（4）细胞在底物上的伪足伸展。通过在无血清培养基中并使用血清预吸附或3T3 SAM包被的盖玻片测量附着情况，可以模拟这些血清效应。对血清预吸附底物的酶处理表明，血清中影响附着的因子对胰蛋白酶非常敏感。底物的血清预吸附以与3T3 SAM包被的效应非常相似的方式刺激了血清培养基中SVT2细胞的附着，而3T3或回复细胞的附着不受影响。平板凝胶电泳分析（十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（PAGE - SDS）凝胶）通过考马斯亮蓝染色鉴定出八种主要血清蛋白：（a）在无细胞情况下与底物结合的；（b）在3T3或SVT2细胞生长至汇合后仍存在于底物上的；这表明在正常或转化细胞生长过程中不会发生主要的分解或血清吸附成分的改变。在3T3或SVT2 SAM电泳图谱中通过放射自显影检测到七种放射性SAM蛋白——其中两种是高分子量成分，对应于先前观察到的葡萄糖胺放射性标记的透明质酸蛋白聚糖；其余五种是SAM中新鉴定的蛋白（其中一种蛋白似乎是肌动蛋白）。3T3和SVT2细胞具有这些七种成分的独特比例。这些数据与以下观点一致，即正常或病毒转化的细胞不是直接附着于培养底物，而是通过特定类别的细胞表面蛋白和多糖的沉积附着于特定类别的底物吸附血清蛋白。
