Collagen contraction mediated by corneal fibroblasts (CFs) is implicated in the maintenance of corneal shape. Given that fibronectin is expressed at sites of corneal stromal wounding, we investigated the effect of fibronectin on CF-mediated collagen gel contraction. Human CFs were cultured in a three-dimensional gel of type I collagen in the absence or presence of various extracellular matrix (ECM) components. The contraction of collagen gels mediated by CFs was evaluated by measurement of changes in gel diameter. The formation of stress fibers and focal adhesions in CFs was examined by fluorescence microscopy. The abundance of paxillin, phosphorylated paxillin, integrins alpha5, beta1, and alpha2, and alpha-smooth muscle actin in CFs was examined by immunoblot analysis. Fibronectin promoted CF-mediated collagen gel contraction in a concentration- and time-dependent manner. Other ECM proteins or glycosaminoglycans did not exhibit such an effect. Fibronectin also induced cell spreading, the formation of stress fibers, and the establishment of focal adhesions containing paxillin in CFs cultured in three-dimensional collagen gels. In addition, it increased the amounts of paxillin, phosphorylated paxillin, and integrins alpha5 and beta1 in these cells. The expression of integrin alpha2 and alpha-smooth muscle actin was not affected by fibronectin, however. Furthermore, the peptide GRGDSP (an antagonist of fibronectin receptors) blocked the stimulatory effect of fibronectin on CF-mediated collagen gel contraction. These results suggest that fibronectin promoted CF-mediated collagen gel contraction in a manner dependent on the formation of stress fibers and focal adhesions, the activation of paxillin, and the up-regulation of integrin alpha5beta1. Fibronectin may therefore contribute to the maintenance of corneal shape by CFs during the healing of stromal wounds.

译文

角膜成纤维细胞 (CFs) 介导的胶原收缩与角膜形状的维持有关。鉴于纤连蛋白在角膜基质损伤部位表达,我们研究了纤连蛋白对CF介导的胶原凝胶收缩的影响。在不存在或存在各种细胞外基质 (ECM) 成分的情况下,在I型胶原蛋白的三维凝胶中培养人CFs。通过测量凝胶直径的变化来评估CFs介导的胶原蛋白凝胶的收缩。通过荧光显微镜检查CFs中应力纤维和粘着斑的形成。通过免疫印迹分析检查CFs中桩蛋白,磷酸化桩蛋白,整联蛋白 α5,β1和 α2以及 α-平滑肌肌动蛋白的丰度。纤连蛋白以浓度和时间依赖性方式促进CF介导的胶原蛋白凝胶收缩。其他ECM蛋白或糖胺聚糖没有表现出这种作用。纤连蛋白还诱导细胞扩散,应力纤维的形成以及在三维胶原蛋白凝胶中培养的CFs中建立含有桩蛋白的粘着斑。此外,它增加了这些细胞中的桩蛋白,磷酸化的桩蛋白以及整合素 α5和 β1的量。然而,整合素 α2和 α-平滑肌肌动蛋白的表达不受纤连蛋白的影响。此外,肽GRGDSP (纤连蛋白受体的拮抗剂) 阻断了纤连蛋白对CF介导的胶原蛋白凝胶收缩的刺激作用。这些结果表明,纤连蛋白以取决于应力纤维和粘着斑的形成,桩蛋白的活化以及整联蛋白alpha5beta1的上调的方式促进CF介导的胶原蛋白凝胶收缩。因此,纤连蛋白可能有助于CFs在基质伤口愈合过程中维持角膜形状。

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