사람 정상 중이점막 상피세포의 섬모세포와 분비세포로의 분화

Abstract

Background & Objectives: Recent technical advances allow serial culture of normal human middle ear epithelial (NHMEE) cells. However, the ciliary differentiation of these cells has not been achieved. The purpose of this study was to establish a culture system to differentiate serially cultured NHMEE cells into ciliated cells. If the ciliated cells were to develop, the percentage of ciliated cells and secretory cells throughout the duration of culture would be measured. We also examined the levels of mucin and lysozyme secretion and their mRNAs in a time-dependent manner.

Materials & Methods: Normal appearing human middle ear mucosa was harvested and subcultured after enzymatic disaggregation. These cells were differentiated in air-liquid interface (ALI) culture for 2 days, 1 week, 2 weeks, 3 weeks and 4 weeks after confluence. On each culture period, the ratio of ciliated cells and secretory cells and the amount of mucin and lysozyme secreted from the cultured cell were measured by immunohistochemical study and dot blotting. The level of mucin genes 5AC (MUC5AC), MUC5B, MUC8 messenger RNA(mRNA)s and the level of lysozyme mRNA were measured on each culture period by reverse transcription (RT)-polymerase chain reaction (PCR).

Results: Ciliogenesis usually started on the 16th-18th day after confluence. The percentage of ciliated cells increased over time up to 10.6% but that of secretory cells remained at about 40% throughout culture duration. By the 14th day after confluence, the amounts of mucin and lysozyme secretion increased rapidly and then maintained a plateau. The expression levels of MUC5B, MUC8 and Lysozyme increased with culture time. Especially, MUC8 showed a dramatic increase on the 28th day after confluence. In contrast, the level of MUC5AC mRNA showed a peak on the 14th day after confluence, and then decreased.

Conclusion: Ciliary differentiation of NHMEE cells can be induced by ALI culture system. Our study also suggests that secretory function develops earlier than ciliogenesis, and that the expressions of MUC5B and MUC8 mRNAs increase as a function of differentiation.