사람 정상중이점막 상피세포의 계대 배양과 분비세포로의 분화 유도

Abstract

BACKGROUND AND OBJECTIVES: The purpose of this study was to subculture normal human middle ear epithelial (NHMEE) cells, investigate whether the subcultured NHMEE cells could have ability to differentiate into secretory cells, and establish a method to get cultured NHMEE cells for further study of human middle ear epithelial differentiation and secretion. MATERIALS AND METHOD: Freshly isolated epithelial cells from healthy middle ear mucosa were subcultured repeatedly after enzymatic disaggregation in serum-free medium on plastic tissue culture dishes. The subcultured cells were counted after every passage and tested for secretory differentiation in air-liquid interface (ALI) cultures. The apical secretion of cultured NHMEE cells were characterized by immunoblotting and Western blotting. RESULTS: Attachment rate of subcultured NHMEE cells was over 70% through every passage. Cells proliferated by 22 fold from passage-1 to passage-2 (P-2), but passage-4 cells did not proliferate. P-2 NHMEE cells in ALI cultures was stained with mucin antibody (H6C5) but not b-tubulin antibody. Cultured NHMEE cells secreted mucin and lysozyme. CONCLUSION: P-2 NHMEE cell cultures retained many important features of normal epithelium and were suitable for conducting many studies of human middle ear epithelial cell biology including cell differentiation and secretion.