Secretory cell differentiation and mucus secretion in cultures of human nasal epithelial cells.

我们开发了一种气液界面培养系统，用于人鼻上皮细胞在确定的无血清培养基中分化为粘液纤毛表型。从鼻息肉中分离出的细胞在胶原凝胶底物上培养。在汇合时，细胞失去了分化细胞的特征，通过去除顶端培养基，只从底部隔间喂养，产生了一个气液界面。在气液界面培养7 d后，观察到分泌细胞和纤毛细胞分化，且分泌细胞数量增加。在第21天，用阿利新蓝-周期性酸-希夫（AB-PAS）或单克隆抗体HCS18对大约一半的上皮细胞进行染色，该单克隆抗体针对上皮分泌（杯状）细胞特异性的人鼻粘蛋白。凹槽印迹分析显示，抗体反应性鼻粘蛋白仅在培养物的鼻尖侧分泌，且分泌量增加，与分泌细胞分化程度一致。上述结果表明，气液界面培养可诱导人鼻上皮细胞的分泌细胞分化，这些细胞分泌的人鼻粘蛋白是鼻上皮分泌（杯状）细胞所特有的。本研究建立的具有抗黏液蛋白单克隆抗体的培养系统可用于研究在各种药理学和生理学操作下人鼻上皮细胞的极化黏液分泌。

We have developed an air-liquid interface culture system for human nasal epithelial cells that differentiate into mucociliary phenotypes in a defined serum-free medium. Dissociated cells obtained from nasal polyp were cultured on a collagen gel substrate. At confluence, the cells lost characteristics of differentiated cells, and an air-liquid interface was produced by removing the apical medium and feeding only from the bottom compartment. After 7 days in an air-liquid interface, secretory cell and ciliated cell differentiation was observed, and the number of secretory cells only increased. At 21 days, about half of the epithelial cells were stained with alcian blue-periodic acid-Schiff (AB-PAS) or an monoclonal antibody HCS18, that was directed against human nasal mucins specific for epithelial secretory (goblet) cells. Slot blot analysis revealed that the antibody-reactive nasal mucins were secreted only the apical side of the cultures, and the amount of secreted mucins increased, coincident with the degree of secretory cell differentiation. These results shows that secretory cell differentiation of human nasal epithelial cells can be induced by air-liquid interface cultures, and these cells secrete human nasal mucins, that are specific for nasal epithelial secretory (goblet) cells. This culture system with an anti-mucin monoclonal antibody developed n this study should be useful for studying polarized mucus secretion from human nasal epithelial cells following various pharmacological and physiological manipulations.