DeBuysscher E, Kennedy J, Mendicino J
In Vitro. 1984 May;20(5):433-46. doi: 10.1007/BF02619589.
Mucus-producing cells were isolated from swine trachea mucosa by a method that included enzymatic digestion of the epithelial surface with Dispase, a neutral protease from Bacillus polymyxa, and differential attachment of the washed cells to culture flasks coated with collagen. Epithelial cells were the major cell type isolated by these procedures. Ciliated cells that did not attach to the flasks were removed by decantation , and fibroblasts were destroyed by the bacterial protease. The isolated cells synthesized respiratory mucins and the rate of secretion was increased about threefold when tracheas were exposed to sulfur dioxide. The cultured cells incorporated both [35S]O4 and [I-14C]N-acetylglucosamine into secreted mucin glycoproteins. The secretion of glycoprotein increased for about 3 d until the cells became confluent, and then a constant rate was observed for a period of at least 7 d. This increase in the output of mucin glycoprotein during the initial 3 d of culture was accompanied by a corresponding increase in the number of mucus-producing cells in the flasks. The results obtained in these and subsequent studies suggest that the rate of formation of mucus-producing cells may be a rate limiting step in the regulation of mucin glycoprotein synthesis in tracheal epithelium. The chemical, physical, and immunological properties of the glycoprotein secreted by isolated tracheal epithelial cells were very similar to the mucin glycoprotein purified from washes of swine trachea epithelium. The purified mucin glycoproteins showed complete cross-reaction with antibodies to trachea mucin glycoprotein. They were eluted near the void volume during gel filtration of Sepharose CL-6B columns. The glycoprotein isolated from culture media under the standard assay conditions had nearly the same carbohydrate composition as samples purified from washes of trachea epithelium. Reduced oligosaccharides released by beta-elimination with dilute alkaline borohydride showed similar elution profiles during chromatography on Bio Gel P-6 columns. Taken collectively, these results suggest that the isolated epithelial cells secreted mucin glycoproteins that were very similar to those synthesized by the intact trachea epithelium under standard incubation conditions.
通过一种方法从猪气管黏膜中分离出产生黏液的细胞，该方法包括用Dispase（一种来自多黏芽孢杆菌的中性蛋白酶）对上皮表面进行酶消化，以及将洗涤后的细胞差异性附着到涂有胶原蛋白的培养瓶上。上皮细胞是通过这些程序分离出的主要细胞类型。未附着在培养瓶上的纤毛细胞通过倾析法去除，成纤维细胞则被细菌蛋白酶破坏。分离出的细胞合成呼吸道黏蛋白，当气管暴露于二氧化硫时，分泌速率增加约三倍。培养的细胞将[35S]O4和[I-14C]N-乙酰葡糖胺都掺入分泌的黏蛋白糖蛋白中。糖蛋白的分泌增加约3天，直到细胞汇合，然后在至少7天的时间内观察到恒定速率。在培养的最初3天中黏蛋白糖蛋白产量的增加伴随着培养瓶中产生黏液的细胞数量相应增加。这些以及后续研究中获得的结果表明，产生黏液的细胞的形成速率可能是气管上皮中黏蛋白糖蛋白合成调节中的一个限速步骤。分离的气管上皮细胞分泌的糖蛋白的化学、物理和免疫学特性与从猪气管上皮洗涤液中纯化的黏蛋白糖蛋白非常相似。纯化的黏蛋白糖蛋白与气管黏蛋白糖蛋白抗体显示出完全交叉反应。在Sepharose CL-6B柱的凝胶过滤过程中，它们在空体积附近被洗脱。在标准测定条件下从培养基中分离的糖蛋白与从气管上皮洗涤液中纯化的样品具有几乎相同的碳水化合物组成。用稀碱性硼氢化钠通过β-消除释放的还原寡糖在Bio Gel P-6柱上的色谱分析中显示出相似的洗脱图谱。总体而言这些结果表明，分离的上皮细胞分泌的黏蛋白糖蛋白与在标准孵育条件下完整气管上皮合成的黏蛋白糖蛋白非常相似。
