Baeza-Squiban A, Boisvieux-Ulrich E, Guilianelli C, Houcine O, Geraud G, Guennou C, Marano F
Laboratoire de Cytophysiologie et Toxicologie cellulaire, Université Paris 7 Denis Diderot, France.
In Vitro Cell Dev Biol Anim. 1994 Jan;30A(1):56-67. doi: 10.1007/BF02631419.
The differentiation of tracheal epithelial cells in primary culture was investigated according to the nature of the extracellular matrix used. Cultures obtained by the explant technique were realized on a type I collagen substratum either as a thin, dried coating or as a thick, hydrated gel supplemented with culture medium and serum. These two types of substratum induced distinct cell morphology and cytokeratin expression in the explant derived cells. Where cells are less proliferating (from Day 7 to 10 of culture), differentiation was evaluated by morphologic ultrastructural observations, immunocytochemical detection of cytokeratins, and determination of cytokeratin pattern by biochemical analysis. The epithelium obtained on gel was multilayered, with small, round basal cells under large, flattened upper cells. The determination of the keratin pattern expressed by cells grown on gel revealed an expression of keratin 13, already considered as a specific marker of squamous metaplasia, that diminished with retinoic acid treatment. Present results demonstrated by confocal microscopy that K13-positive cells were large upper cells with a dense keratin network, whereas lower cells were positively stained with a specific monoclonal antibody to basal cells (KB37). Moreover, keratin neosynthesis analysis pointed out a higher expression of K6, a marker of hyperproliferation, on gel than on coating. All these data suggest a differentiation of rabbit tracheal epithelial cells grown on gel toward squamous metaplasia. By contrast, the epithelium observed on coating is nearly a monolayer of very large and spread out cells. No K13-positive cells were observed, but an increase in the synthesis of simple epithelium marker (K18) was detected. These two substrata, similar in composition and different in structure, induce separate differentiation and appear as good tools to explore the mechanisms of differentiation of epithelial tracheal cells.
根据所使用的细胞外基质的性质，对原代培养的气管上皮细胞的分化进行了研究。通过外植体技术获得的培养物在I型胶原基质上实现，该基质可以是薄的、干燥的涂层，也可以是补充了培养基和血清的厚的、水合凝胶。这两种类型的基质在外植体衍生细胞中诱导出不同的细胞形态和细胞角蛋白表达。在细胞增殖较少的情况下（培养第7天至10天），通过形态学超微结构观察、细胞角蛋白的免疫细胞化学检测以及生化分析确定细胞角蛋白模式来评估分化。在凝胶上获得的上皮是多层的，大的扁平上层细胞下方是小的圆形基底细胞。对在凝胶上生长的细胞所表达的角蛋白模式的测定显示，角蛋白13表达，角蛋白13已被视为鳞状化生的特异性标志物，其表达随着视黄酸处理而减少。共聚焦显微镜显示的当前结果表明，K13阳性细胞是具有致密角蛋白网络的大的上层细胞，而较低层细胞用基底细胞特异性单克隆抗体（KB37）呈阳性染色。此外，角蛋白新合成分析指出，凝胶上增殖标志物K6的表达高于涂层上的表达。所有这些数据表明，在凝胶上生长的兔气管上皮细胞向鳞状化生分化。相比之下，在涂层上观察到的上皮几乎是单层的非常大且伸展的细胞。未观察到K13阳性细胞，但检测到简单上皮标志物（K18）合成增加。这两种基质组成相似但结构不同，诱导不同的分化，似乎是探索气管上皮细胞分化机制的良好工具。
