人鼻腔纤毛上皮细胞的浸没培养  

Submerged culture of human nasal ciliated epithelial cells

作　　者：矫健[1,2] 孟娜[1,2] 张罗[1,2] 

机构地区：[1]首都医科大学附属北京同仁医院耳鼻咽喉头颈外科,北京100730 [2]北京市耳鼻咽喉科研究所教育部耳鼻咽喉科学重点实验室

出　　处：《临床耳鼻咽喉头颈外科杂志》2014年第12期898-902,共5页Journal of Clinical Otorhinolaryngology Head And Neck Surgery

基　　金：国家杰出青年科学基金(No:81025007);国家自然科学基金(No:81100704);北京市自然科学基金重点项目(No:7131006);北京市科技新星计划(No:Z111107054511061);高等学校博士学科点专项科研基金(No:20111107120004);首都卫生发展科研专项(No:2011-1017-03);卫生部2012年度公益性行业科研专项(No:201202005);北京市卫生系统高层次卫生技术人才学科带头人基金(No:2009-02-007)

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7～10d达到高峰后逐渐减少，纤毛细胞存活时间维持在14～21d；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100μmol／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。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 ceils by high-speed digital microscopic imaging system. Result： （1）Under 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; （2）Under 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; （3）At day 7, Immunofluo- rescence of β-tubulin Ⅳ and ZO-1 showed a good confluence and differentiation of cilia, and the percentage of cilia ted epithelial cells accounted for 20%- 30% ; （4）Basal 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;（5）ATP, 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.

关 键 词：人 鼻腔 纤毛 上皮细胞 细胞培养 

分 类 号：R765[医药卫生—耳鼻咽喉科]