Interaction of fibronectin-coated beads with CHO cells.
Interaction of fibronectin-coated beads with CHO cells.
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纤连蛋白包被的珠子与 CHO 细胞的相互作用。
DOI：
10.1016/0014-4827(84)90632-3
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发表时间：
1984
影响因子：
3.7
通讯作者：
Juliano,RL
中科院分区：
文献类型：
--
作者：
Schwarz,MA;Juliano,RL
成纤维细胞粘附至基质的最早事件之一是细胞对大分子粘附因子（例如纤连蛋白）的识别。这一早期事件之后是一系列复杂的细胞改变，导致粘附和扩散。为了鉴定参与初始细胞纤连蛋白识别步骤的细胞表面成分，我们采用了一种涉及涂有放射性标记血浆纤连蛋白（Fn）的乳胶颗粒的测定方法。在该实验室之前的研究中（Harper & Juliano, J cell biol 87 (1980) 755）[28]，我们证明 Fn 介导的 CHO 细胞粘附具有温度依赖性、阳离子依赖性并且对细胞骨架破坏剂敏感；相反， 3 H-Fn珠的结合不受这些因素的影响，表明该过程反映了细胞表面的结合和识别事件，其独立于细胞骨架和代谢活性。 3 H-Fn珠与细胞结合的生物特异性通过抗Fn抗血清完全阻断该过程的能力得到证实。为了检查可能介导结合的表面成分，我们在与细胞孵育之前用纯化的糖胺聚糖 (GAG) 或糖脂处理 Fn 珠。在测试的 GAG 中，肝素、硫酸乙酰肝素和硫酸皮肤素以剂量相关的方式阻断珠子结合，其中肝素最有效。神经节苷脂 GT1 和 GM1 也抑制珠子结合。然而，用神经氨酸酶处理细胞对珠子结合没有影响，而随后通过薄层色谱对处理的细胞进行的分析显示，GM3（主要的 CHO 细胞神经节苷脂）的量急剧减少。 CHO 细胞还与一组蛋白水解酶一起孵育，以研究细胞表面蛋白或糖蛋白在 Fn 珠结合中的可能作用。我们发现 3 H-Fn 珠结合对嗜热菌蛋白酶、链霉蛋白酶和木瓜蛋白酶的预处理非常敏感，但对胰蛋白酶的处理仅中度敏感。根据我们的研究结果，我们建议：(1) Fn 珠与 CHO 细胞的结合反映了粘附过程的早期步骤； (2) 糖脂可能会阻碍珠子结合，但可能不是 Fn 的内源性结合位点； (3) 蛋白酶敏感成分（糖蛋白或蛋白聚糖）可能更有可能作为 Fn 的细胞表面结合位点。
One of the earliest events in the adhesion of fibroblasts to a substratum is the recognition by the cells of macromolecular adhesive factors, such as fibronectin. This early event is followed by a complex series of cell alterations leading to adhesion and spreading. To identify cell surface components involved in the initial cell-fibronectin recognition step, we have employed an assay involving latex particles coated with radiolabelled plasma Fibronectin (Fn). In previous studies from this laboratory (Harper & Juliano, J cell biol 87 (1980) 755) [28], we demonstrated that Fn-mediated adhesion of CHO cells is temperature-dependent, cation-dependent and sensitive to cytoskeletal disrupting agents; by contrast, binding of3H-Fn beads was unaffected by these factors, indicating that this process reflects binding and recognition events at the cell surface which are independent of cytoskeletal and metabolic activity. Biological specificity of3H-Fn bead-to-cell binding was confirmed by the ability of anti-Fn antisera to completely block the process. To examine surface components which may mediate binding we treated Fn beads with purified glycosaminoglycans (GAGs) or glycolipids prior to incubation with cells. Among the GAGs tested, heparin, heparan sulfate and dermatan sulfate blocked bead binding in a dose-related fashion with heparin being most potent. The gangliosides GT1, and GM1, also inhibited bead binding. However, treatment of cells with neuraminidase had no effect on bead binding while subsequent analysis of treated cells by thin layer chromatography revealed a drastic reduction in the amount of GM3, the predominant CHO cell ganglioside. CHO cells were also incubated with a panel of proteolytic enzymes to study the possible role of cell surface proteins or glycoproteins in Fn bead binding. We found3H-Fn bead binding to be quite sensitive to pretreatment with thermolysin, pronase, and papain but only moderately sensitive to treatment with trypsin. From our findings we suggest: (1) binding of Fn beads to CHO cells reflects an early step in the adhesion process; (2) glycolipids may block bead binding but are probably not the endogenous binding site for Fn; (3) protease sensitive components (glycoproteins or proteoglycans) may be more likely candidates as cell surface-binding sites for Fn.