Venaille T J, Mendis A H, Warton A, Walker L, Papadimitriou J M, Robinson B W
University Department of Medicine, Queen Elizabeth II Medical Centre, Nedlands, WA, Australia.
Immunol Cell Biol. 1989 Dec;67 ( Pt 6):359-69. doi: 10.1038/icb.1989.52.
Detachment of epithelial structures from underlying basement membrane (BM) represents an important component of a number of human disease processes e.g. airway and alveolar diseases, gastrointestinal ulceration, and retinal diseases. This study describes a method of evaluating human epithelial cell detachment from BM that is simple, rapid, inexpensive, quantifiable and which, because it utilizes BM rather than tissue culture plastic, more closely mimics the in vivo situation than other methods. In this model human amnionic epithelial cells attached to their underlying BM are isolated from fresh placentae and mounted in a multi-well chemotaxis assembly. These membranes can be studied with the epithelial cell monolayer intact. Protease-induced detachment of the epithelial cells from the underlying BM was readily quantifiable using light microscopy and spectroscopy. Following removal of the native amnionic epithelial cells, immunoperoxidase staining for the BM attachment proteins laminin, fibronectin, and type IV collagen demonstrated that these molecules remain intact. The BM could also be used as an attachment surface to reconstitute other epithelial cell monolayers. Cultured human amnionic cells and human respiratory epithelial cells were both able to attach to the denuded BM in the absence of serum (% attachment = 85 +/- 15% and 92 +/- 8% respectively, P = 0.8). Natural BM was a better substrate for epithelial cell attachment than tissue culture plastic in that, in the absence of serum, cultured epithelial cell attachment to tissue culture plastic was 20 +/- 4% of the value for BM (P less than 0.05). Furthermore, cells attached to plastic adhered less effectively than to BM in that trypsin concentrations required to induce 50% cell detachment were 0.72 +/- 0.4 for plastic and 62 +/- 13 BAEE U/ml for BM (P less than 0.001). In view of the complex protein interactions known to be involved in the anchorage of human epithelial cells to BM, it is likely this model will be a useful tool for evaluating the mechanisms underlying human epithelial cell attachment and detachment in a variety of normal and disease situations.
上皮结构与下方基底膜（BM）分离是许多人类疾病过程的重要组成部分，例如气道和肺泡疾病、胃肠道溃疡以及视网膜疾病。本研究描述了一种评估人上皮细胞与基底膜分离的方法，该方法简单、快速、廉价、可量化，并且由于它使用的是基底膜而非组织培养塑料，因此比其他方法更能紧密模拟体内情况。在这个模型中，将附着于其下方基底膜的人羊膜上皮细胞从新鲜胎盘中分离出来，并安装在多孔趋化性组件中。这些膜可以在完整的上皮细胞单层状态下进行研究。使用光学显微镜和光谱法可以很容易地对蛋白酶诱导的上皮细胞从下方基底膜的分离进行量化。去除天然羊膜上皮细胞后，对基底膜附着蛋白层粘连蛋白、纤连蛋白和IV型胶原进行免疫过氧化物酶染色，结果表明这些分子保持完整。基底膜还可以用作附着表面来重建其他上皮细胞单层。培养的人羊膜细胞和人呼吸道上皮细胞在无血清情况下都能够附着于裸露的基底膜（附着率分别为85±15%和92±8%，P = 0.8）。天然基底膜作为上皮细胞附着的底物比组织培养塑料更好，因为在无血清情况下，培养的上皮细胞在组织培养塑料上的附着率是在基底膜上附着率的20±4%（P＜0.05）。此外，附着于塑料的细胞比附着于基底膜的细胞黏附效果差，因为诱导50%细胞分离所需的胰蛋白酶浓度，对于塑料为0.72±0.4，对于基底膜为62±13 BAEE U/ml（P＜0.001）。鉴于已知人类上皮细胞与基底膜的锚定涉及复杂的蛋白质相互作用，这个模型很可能成为评估各种正常和疾病情况下人类上皮细胞附着和分离机制的有用工具。
