Moon S K, Lim D J, Lee H K, Kim H N, Yoo J H
Depatment of Cell Biology, House Ear Institute, Los Angeles, California, USA.
Acta Otolaryngol. 2000 Oct;120(8):933-9. doi: 10.1080/00016480050218654.
For the advanced study of the cell and molecular biology of middle ear mucosa, an in vitro cell culture system is required. Although middle ear epithelial cells have been cultured from various species of laboratory animal, there have been no reports concerning a serial subculture system of human middle ear epithelial cells. In this paper, we describe the establishment of a primary culture system of human middle ear epithelial cells using a serum-free conditioned medium and the characterization of these cells by the expression of phenotypic characteristics of epithelial cells and mucin genes. Cultured cells were anchorage-dependent in terms of growth and showed a polygonal cobblestone-like appearance: desmosomes in the cell junction were observed by electron microscopy. In the immunocytochemical study, cytokeratin (epithelial cell marker) was expressed in all cultured cells. but von Willebrand factor (endothelial cell marker) was not. Unexpectedly, vimentin (fibroblast marker) was locally expressed, and a double stain showed the co-expression of both cytokeratin and vimentin in the same cell. The products of reverse transcriptase polymerase chain reaction from cultured cells yielded distinct bands compatible with the expected sizes of the MUC1, MUC2, MUC5AC and MUC5B genes. This culture system will allow us to prepare the cell line and to perform advanced studies of human middle ear mucosal biology.
为了深入研究中耳黏膜的细胞和分子生物学，需要一个体外细胞培养系统。尽管已经从多种实验动物中培养出了中耳上皮细胞，但尚未有关于人中耳上皮细胞连续传代培养系统的报道。在本文中，我们描述了使用无血清条件培养基建立人中耳上皮细胞原代培养系统的方法，并通过上皮细胞表型特征和黏蛋白基因的表达对这些细胞进行了表征。培养的细胞在生长方面依赖贴壁，呈现多边形鹅卵石样外观：通过电子显微镜观察到细胞连接处有桥粒。在免疫细胞化学研究中，细胞角蛋白（上皮细胞标志物）在所有培养细胞中均有表达，但血管性血友病因子（内皮细胞标志物）未表达。出乎意料的是，波形蛋白（成纤维细胞标志物）有局部表达，双重染色显示同一细胞中细胞角蛋白和波形蛋白共表达。培养细胞的逆转录聚合酶链反应产物产生了与预期大小的MUC1、MUC2、MUC5AC和MUC5B基因相符的清晰条带。该培养系统将使我们能够制备细胞系并对人中耳黏膜生物学进行深入研究。
