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摘要: 目的:浸没培养法培养人鼻腔纤毛上皮细胞,为纤毛相关研究及经鼻药物安全性评价等研究提供可靠的细胞学模型。方法:采用低温酶消化法,浸没培养人鼻腔纤毛上皮细胞,倒置相差显微镜观察细胞生长状况,扫描电镜及免疫细胞化学方法观察细胞融合及纤毛分化状态,高速数字化显微视频成像系统检测纤毛摆动频率。结果:①相差显微镜下,纤毛细胞数量逐渐增加,至7~10 d达到高峰后逐渐减少,纤毛细胞存活时间维持在14~21 d;②细胞培养第7天,扫描电镜可见鼻腔上皮细胞表面覆盖纤毛或微绒毛,杯状细胞及无纤毛柱状上皮细胞相间排列;③细胞培养第7天,Ⅳ型β-微管蛋白、闭合小环蛋白-1免疫荧光结果显示细胞融合及纤毛分化良好,纤毛细胞比例可达20%~30%;④培养第7、14、21天纤毛摆动基础频率分别为(10.73±2.15)Hz、(9.92±1.97)Hz、(10.30±2.11)Hz,无明显统计学差异;⑤外源性刺激剂100 μmol/L ATP可明显增加纤毛摆动频率。结论:应用酶消化法浸没培养人鼻腔纤毛上皮细胞,细胞融合及纤毛分化状态良好,纤毛摆动活跃,对外源性刺激反应灵敏,是应用于纤毛相关研究及经鼻药物安全性评价等研究的一个较为理想的细胞模型。Abstract: Objective: To culture human nasal ciliated epithelial cells in a submerged fashion so as to establish a reliable cell culture model for research about cilia and safety evaluation of intranasal drugs.Method: We cultured the human nasal ciliated epithelial cells by low-temperature enzymatic digestion in a submerged fashion. And we observed the cell growth state under the inverted phase-contrast microscopy and the confluence and differentiation of ciliated cells by scanning electron microscopy and immunocytochemistry. At last we measured the ciliary beat frequency (CBF) of cultured epithelial cells by high-speed digital microscopic imaging system.Result: ①Under phase contrast microscopy, the number of ciliated cells increased with time, reached the maximum at 7-10 day, then decreased, and the survival time of ciliated epithelial cells maintained for 14-21 d; ②Under scanning electron microscopy at day 7, the cilia and small microvilli were observed to be covered on the surface of nasal ciliated epithelial cells at, and goblet cells and nonciliated columnar cells arranged alternately; ③At day 7, Immunofluorescence of β-tubulin IV and ZO-1 showed a good confluence and differentiation of cilia, and the percentage of ciliated epithelial cells accounted for 20%-30%;④Basal CBF of cultured epithelial cells was (10.73±2.15)Hz, (9.92±1.97)Hz, (10.30±2.11)Hz at day 7, 14 and 21, respectively, which showed no significant difference among them;⑤ATP, an exogenous stimulating agent, significantly increased the CBF of cultured epithelial cells at the concentration of 100 μmol/L.Conclusion: Human nasal epithelial cells cultured with enzymatic digestion in a submerged fashion manifest with good confluence and differentiation status, active cilia beat and sensitive response to exogenous stimuli. Therefore, it may serve as an ideal cell model for cilia-related research and safety evaluation of intranasal drugs.
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Key words:
- human /
- nasal cavity /
- cilia /
- epithelial cells /
- cell culture
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