Hérard A L, Pierrot D, Hinnrasky J, Kaplan H, Sheppard D, Puchelle E, Zahm J M
Institut National de la Santé et de la Recherche Médicale U314, Reims France.
Am J Physiol. 1996 Nov;271(5 Pt 1):L726-33. doi: 10.1152/ajplung.1996.271.5.L726.
The cell migration that occurs during wound repair is dependent on modifications of the cell-matrix interaction in which extracellular matrix proteins and their receptors, the integrins, are involved. To study the interactions between airway epithelial cells and the extracellular matrix during the process of wound repair, we developed an in vitro wound model of human epithelial cells. Surface epithelial cells were dissociated from human nasal polyps and cultured on a type I collagen matrix. At confluency, a wound was made by the addition of 2 microliters of NaOH (1 N) to the cell culture. After the cell culture was washed, the wound area was recorded every 12 h for 96 h by a videomicroscopic technique. We calculated the wound-repair index that represents the decrease in the wound area per hour. Using immunofluorescence techniques, we first examined the localization, during wound repair, of fibronectin and of the beta 1-, alpha v-, alpha 2-, alpha 3-, and alpha 5-integrin subunits. Secondly, we carried out a series of wound-repair blocking experiments with the use of anti-integrin or anti-fibronectin antibodies diluted in the culture medium. We observed that fibronectin and the alpha 5- integrin subunit were exclusively expressed by the migratory cells in the wounded area. No difference in the localization of the alpha v-, alpha 2-, and alpha 3-integrin subunits was observed between the nonrepairing and repairing cells. The blocking experiments showed a significant decrease in the wound-repair index in the presence of either the anti-beta 1, -alpha 3, alpha 5, or the anti-fibronectin antibodies. Furthermore, the addition of fibronectin to the culture medium induced a significant increase in the wound repair index. These results suggest that fibronectin and the corresponding alpha 5 beta 1-integrin play an important role in the process of airway epithelium wound repair.
伤口修复过程中发生的细胞迁移依赖于细胞与基质相互作用的改变，其中涉及细胞外基质蛋白及其受体整合素。为了研究伤口修复过程中气道上皮细胞与细胞外基质之间的相互作用，我们建立了人上皮细胞的体外伤口模型。从人鼻息肉中分离出表面上皮细胞，并在I型胶原基质上培养。细胞汇合后，通过向细胞培养物中加入2微升NaOH（1N）制造伤口。细胞培养物洗涤后，通过视频显微镜技术每12小时记录一次伤口面积，持续96小时。我们计算了代表每小时伤口面积减少的伤口修复指数。使用免疫荧光技术，我们首先检查了伤口修复过程中纤连蛋白以及β1-、αv-、α2-、α3-和α5-整合素亚基的定位。其次，我们使用在培养基中稀释的抗整合素或抗纤连蛋白抗体进行了一系列伤口修复阻断实验。我们观察到，纤连蛋白和α5-整合素亚基仅由伤口区域的迁移细胞表达。在未修复细胞和修复细胞之间未观察到αv-、α2-和α3-整合素亚基定位的差异。阻断实验表明，在存在抗β1、-α3、α5或抗纤连蛋白抗体的情况下，伤口修复指数显著降低。此外，向培养基中添加纤连蛋白可导致伤口修复指数显著增加。这些结果表明，纤连蛋白和相应的α5β1-整合素在气道上皮伤口修复过程中起重要作用。
