Verma M, Sanadi A R, Davidson E A
Department of Biochemistry, Georgetown University Medical Center, Washington, DC 20007, USA.
Glycobiology. 1994 Dec;4(6):825-36. doi: 10.1093/glycob/4.6.825.
To study the regulation of mucin synthesis in canine tracheal epithelial cells, it is desirable to establish a cell line which synthesizes mucin continuously. We adopted the approach of immortalizing canine tracheal epithelial cells using a vector encoding the human papillomavirus (type 18) E6 and E7 genes. The E6 and E7 genes are essential and sufficient for the immortalization of human genital keratinocytes, as well as human tracheal epithelium. Primary epithelial cells from dog trachea were transfected with a vector containing HPV18 genes E6 and E7. The resultant cells (CT1) were cloned and maintained in selective medium supplemented with growth factors and hormones. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicated the expression of the canine tracheal mucin (CTM) mRNA in these cells. The half-life of the CTM mRNA was found to be 45-60 min. Incorporation of labelled precursor (glucosamine) indicated that high-molecular-weight mucin glycoprotein was synthesized by these immortalized cells, which reacted with the antiserum to the native CTM. Equilibrium gradient centrifugation analysis showed that the buoyant density of the mucin synthesized in CT1 cells (1.486 g/ml) was similar to the reported value for native CTM (1.5 g/ml). Mucin which was isolated from immortalized cells was not a proteoglycan as chondroitinase treatment had no effect. These results suggest that CT1 cells synthesize a mucin glycoprotein which exhibits properties similar to native CTM. When characterized by immunostaining with a pool of monoclonal antibodies, these cells showed common epithelial antigens related to keratin expression. The CT1 cell line represents a unique resource for studying mucin biosynthesis and regulation.
为了研究犬气管上皮细胞中黏蛋白合成的调控机制，建立一个能持续合成黏蛋白的细胞系是很有必要的。我们采用了一种方法，即利用编码人乳头瘤病毒（18型）E6和E7基因的载体使犬气管上皮细胞永生化。E6和E7基因对于人生殖器角质形成细胞以及人气管上皮的永生化是必不可少且足够的。用含有HPV18基因E6和E7的载体转染来自犬气管的原代上皮细胞。将所得细胞（CT1）进行克隆，并在补充了生长因子和激素的选择性培养基中培养。逆转录聚合酶链反应（RT-PCR）分析表明这些细胞中存在犬气管黏蛋白（CTM）mRNA的表达。发现CTM mRNA的半衰期为45 - 60分钟。掺入标记前体（葡糖胺）表明这些永生化细胞合成了高分子量的黏蛋白糖蛋白，其与抗天然CTM的抗血清发生反应。平衡梯度离心分析表明CT1细胞中合成的黏蛋白的浮力密度（1.486 g/ml）与报道的天然CTM的值（1.5 g/ml）相似。从永生化细胞中分离出的黏蛋白不是蛋白聚糖，因为软骨素酶处理没有效果。这些结果表明CT1细胞合成了一种黏蛋白糖蛋白，其性质与天然CTM相似。当用一组单克隆抗体进行免疫染色鉴定时，这些细胞显示出与角蛋白表达相关的常见上皮抗原。CT1细胞系是研究黏蛋白生物合成和调控的独特资源。
