Gray T E, Guzman K, Davis C W, Abdullah L H, Nettesheim P
Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.
Am J Respir Cell Mol Biol. 1996 Jan;14(1):104-12. doi: 10.1165/ajrcmb.14.1.8534481.
The goal of our studies was to establish procedures for subculturing normal human tracheobronchial epithelial (NHTBE) cells without compromising their ability to differentiate into mucous and ciliated cells (i.e., differentiation competence) and to study the regulation of airway secretions by epidermal growth factor (EGF) and retinoic acid (RA). Primary NHTBE cells were obtained from a commercial source and subcultured repeatedly in serum-free medium on plastic tissue culture dishes. The subcultured cells were tested after every passage for differentiation competence in air-liquid interface (ALI) cultures. The apical secretions of cultured NHTBE cells were characterized by immunoblotting, Western blotting, or enzyme-linked immunosorbent assay using a variety of antibodies. They contained mucin-like materials as well as lysozyme, lactoferrin, and secretory leukocyte protease inhibitor (SLPI). We found that an EGF concentration of 25 ng/ml, which is commonly used in airway cell cultures, adversely affected growth, mucin production, and morphology of ALI cultures and that RA was essential for mucociliary differentiation. Without RA, the epithelium became squamous and mucin secretions decreased 300- to 900-fold. In contrast, secretion of lysozyme, lactoferrin, and SLPI was significantly increased in RA-depleted cultures. Cells of passage 2 (P-2) through P-4 remained competent to differentiate into mucous and ciliated cells when grown in ALI cultures. However, mucin secretion and ciliagenesis decreased in P-3 and P-4 cell cultures and P-3 but not P-4 cell cultures exhibited bioelectric properties characteristic of airway epithelium. We concluded that P-2 and P-3 NHTBE cell cultures retain many important features of normal airway epithelium. This enables one to conduct many studies of airway cell biology with a greatly expanded (6,000-fold) cell pool.
我们研究的目标是建立正常人气管支气管上皮（NHTBE）细胞传代培养的程序，同时不损害其分化为黏液细胞和纤毛细胞的能力（即分化能力），并研究表皮生长因子（EGF）和视黄酸（RA）对气道分泌物的调节作用。原代NHTBE细胞购自商业来源，并在塑料组织培养皿中的无血清培养基中反复传代培养。每次传代后，对传代培养的细胞进行气液界面（ALI）培养中的分化能力测试。使用多种抗体，通过免疫印迹、蛋白质印迹或酶联免疫吸附测定对培养的NHTBE细胞的顶端分泌物进行表征。它们含有黏蛋白样物质以及溶菌酶、乳铁蛋白和分泌型白细胞蛋白酶抑制剂（SLPI）。我们发现，气道细胞培养中常用的25 ng/ml的EGF浓度对ALI培养物的生长、黏蛋白产生和形态有不利影响，并且RA对于黏液纤毛分化至关重要。没有RA，上皮会变成鳞状，黏蛋白分泌减少300至900倍。相比之下，在缺乏RA的培养物中，溶菌酶、乳铁蛋白和SLPI的分泌显著增加。第2代（P-2）至第4代细胞在ALI培养中生长时仍有能力分化为黏液细胞和纤毛细胞。然而，P-3和P-4细胞培养物中的黏蛋白分泌和纤毛形成减少，并且P-3细胞培养物（而非P-4细胞培养物）表现出气道上皮特有的生物电特性。我们得出结论，P-2和P-3 NHTBE细胞培养物保留了正常气道上皮的许多重要特征。这使得人们能够利用大大扩大（6000倍）的细胞库进行许多气道细胞生物学研究。
