Wu R, Yankaskas J, Cheng E, Knowles M R, Boucher R
Am Rev Respir Dis. 1985 Aug;132(2):311-20. doi: 10.1164/arrd.1985.132.2.311.
Ham's F12 medium supplemented with insulin (Ins), transferrin (Tf), epidermal growth factor (EGF), hydrocortisone (HC), T3, cholera toxin (CT), and bovine hypothalamus extract (BHE) was developed for in vitro growth of human nasal epithelial (HNE) cells. The HNE cells were dissociated from freshly excised nasal polyps or turbinates with protease. Colony-forming efficiency of primary HNE cells was approximately 5%. Growth studies showed Ins, BHE, and CT were essential for growth; HC, EGF, Tf, and T3 were also stimulatory for growth. The growth rate in this serum-free, hormone-supplemented medium was 24 h per population doubling. Up to 20 population doublings and 3 passages of dissociated HNE cells could be achieved. Addition of serum to this culture medium inhibited epithelial cell growth. Vitamin A had no apparent effect on cell growth but induced an alteration in the morphologic characteristics of the cell. The epithelial nature of cultured cells was confirmed by positive staining with antihuman keratin antibody, ultrastructural studies, and by formation of a columnar, ciliated epithelium in denuded tracheal grafts repopulated by these cultured HNE cells. Biochemical analyses of glycoproteins (labeled with 3H-glucosamine and/or 35S-sulfate) secreted by cultured HNE cells were unable to demonstrate the secretion of mucinlike glycoproteins in culture. Instead, major secretory products of cultured cells were hyaluronate and heparan sulfate. These results were in agreement with morphologic observations that showed no mucus-secreting granules in cultured cells. Dome formation was observed in high cell density cultures. We conclude that HNE cells can be cultured in well-defined culture media. As indicated by formation of domes, these cells may be useful for in vitro ion transport studies. Further differentiation, however, may be required for studies of mucin synthesis.
添加胰岛素（Ins）、转铁蛋白（Tf）、表皮生长因子（EGF）、氢化可的松（HC）、三碘甲状腺原氨酸（T3）、霍乱毒素（CT）和牛下丘脑提取物（BHE）的Ham's F12培养基被开发用于人鼻上皮（HNE）细胞的体外生长。HNE细胞用蛋白酶从新鲜切除的鼻息肉或鼻甲中分离出来。原代HNE细胞的集落形成效率约为5%。生长研究表明，Ins、BHE和CT对生长至关重要；HC、EGF、Tf和T3也对生长有促进作用。在这种无血清、添加激素的培养基中，细胞群体倍增时间为24小时。解离的HNE细胞最多可实现20次群体倍增和传代3次。向该培养基中添加血清会抑制上皮细胞生长。维生素A对细胞生长无明显影响，但会诱导细胞形态特征发生改变。通过抗人角蛋白抗体阳性染色、超微结构研究以及用这些培养的HNE细胞重新填充的去上皮气管移植物中形成柱状纤毛上皮，证实了培养细胞的上皮性质。对培养的HNE细胞分泌的糖蛋白（用3H-葡萄糖胺和/或35S-硫酸盐标记）进行生化分析，未能证明培养物中分泌了黏蛋白样糖蛋白。相反，培养细胞的主要分泌产物是透明质酸和硫酸乙酰肝素。这些结果与形态学观察结果一致，即培养细胞中未观察到分泌黏液的颗粒。在高细胞密度培养物中观察到穹顶形成。我们得出结论，HNE细胞可以在成分明确的培养基中培养。如穹顶的形成所示，这些细胞可能有助于体外离子转运研究。然而，对于黏蛋白合成的研究可能需要进一步分化。
