Gibson W T, Scott I R, Saunders H J, Brunskill J E, Harding C R
Eur J Cell Biol. 1984 Jan;33(1):75-83.
A broad range of analytical methods has been used to investigate the expression of key differentiation markers in keratinocytes cultured by a modified feeder layer technique. Cultures were stratified and showed many of the features characteristic of epidermal differentiation in vivo including tonofilaments, desmosomes, loss of organelles and thickening of the plasma membrane to form the cornified envelope. Profilaggrin synthesis was detected by 32P-incorporation and the presence of filaggrin suggested that it was broken down by the normal route. Staining with the lectin from Ulex europeus revealed the presence of a fucose-containing cell-surface glycoprotein. Keratin synthesis was shown by 3H-leucine incorporation and keratins were analysed by two-dimensional gel electrophoresis in comparison with those from different levels of the epidermis. Quantitative and qualitative differences were found between in vivo and in vitro epidermal differentiation. In particular, cornified envelope numbers were low, in keeping with the observation by electron microscopy of only one layer of cells with this structure. The absence of a true stratum corneum in vitro was also indicated by the virtual absence of histidase activity and stratum corneum keratins. The keratin species present in vitro most closely resembled those of the basal cells of the epidermis, although even in this case differences were observed. The evidence as a whole is consistent with the belief that epidermal cells do synthesise in vitro many of the important proteins involved in differentiation, but that they nevertheless do not develop a true keratinised stratum corneum.
一系列广泛的分析方法已被用于研究通过改良饲养层技术培养的角质形成细胞中关键分化标志物的表达。培养物呈现分层，显示出许多体内表皮分化的特征，包括张力丝、桥粒、细胞器丧失以及质膜增厚以形成角质化包膜。通过掺入³²P检测前丝聚合蛋白的合成，而丝聚合蛋白的存在表明其通过正常途径被分解。用欧洲荆豆凝集素染色显示存在一种含岩藻糖的细胞表面糖蛋白。通过掺入³H-亮氨酸显示角蛋白合成，并通过二维凝胶电泳分析角蛋白，与来自表皮不同层次的角蛋白进行比较。发现体内和体外表皮分化存在定量和定性差异。特别是，角质化包膜数量较低，这与电子显微镜观察到只有一层具有这种结构的细胞的结果一致。体外几乎不存在组织蛋白酶活性和角质层角蛋白也表明不存在真正的角质层。体外存在的角蛋白种类与表皮基底细胞的角蛋白种类最为相似，尽管即便如此仍观察到差异。总体证据与以下观点一致：表皮细胞在体外确实合成了许多参与分化的重要蛋白质，但它们仍然无法形成真正的角质化角质层。
