人鼻息肉上皮细胞的气液界面培养  

Culture of human nasal polyp epithelial cells at an air-liquid surface

作　　者：孟娜[1] 矫健[1] 张罗[1] 

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市耳鼻咽喉科研究所教育部耳鼻咽喉科学重点实验室(首都医科大学),100730

出　　处：《中华耳鼻咽喉头颈外科杂志》2014年第1期49-53,共5页Chinese Journal of Otorhinolaryngology Head and Neck Surgery

基　　金：国家杰出青年科学基金（81025007）;国家自然科学基金（81100704）;北京市自然科学基金重点项目（7131006）;北京市科技新星计划（z111107054511061）;高等学校博士学科点专项科研基金（20111107120004）;首都卫生发展科研专项（2011-1017-03）;卫生部2012年度公益性行业科研专项（201202005）;北京市卫生系统高层次卫生技术人才学科带头人基金（2009-02-007）

摘　　要：目的气液界面培养人鼻腔纤毛上皮细胞，为深入研究鼻腔黏液纤毛传输系统提供良好的细胞模型。方法采用低温酶消化法，气液界面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，扫描电镜和免疫细胞化学法观察细胞增殖、融合及分化情况，高速数字显微视频成像系统检测纤毛摆动频率及对外源性刺激剂三磷酸腺苷（adenosinetriphosphate，ATP）的反应性。采用Prism4．0软件进行数据分析。结果光镜下Transwell支持膜上细胞24h贴壁生长良好，浸没培养约1周后，单层细胞融合达80％-90％，细胞间衔接紧密成铺路石状结构，此时建立气液界面培养。气液界面培养7d，扫描电镜可见鼻腔上皮细胞表面覆盖纤毛或微绒毛，纤毛细胞分化良好，成簇状分布，杯状细胞及无纤毛柱状上皮细胞相间排列。气液界面培养14d，IV型β-微管蛋白（p—tubulinIV），闭合小环蛋白-1（Zonaoccludens-1，ZO-1）免疫荧光结果显示细胞融合及纤毛上皮细胞分化良好，纤毛细胞比例可达50％～60％。气液界面培养7、14、21、28、35d，上皮细胞纤毛摆动基础频率分别为（8．42±1．24）、（8．71±1．11）、（9．17±1．11）、（8．89±0．91）、（8．99±0．91）Hz，差异无统计学意义（F=1．451，P〉0．05）。外源性刺激剂100μmol／LATP可明显增加纤毛摆动频率。结论低温酶消化法，气液界面培养模式下可获得分化良好的人鼻腔纤毛上皮细胞，其形态和功能与体内接近，且能较长时间维持其正常形态及功能，可以为深入研究黏液纤毛传输系统提供细胞分化模型。Objective To study the human nasal ciliated epithelial cells at an air-liquid surface （ALI） so as to establish a reliable cell culture model for nasal mucociliary transport study. Methods The human nasal ciliated epithelial cells were cuhured by low-temperature enzymatic digestion method at an air- liquid surface, the cell growth behavior was observed under the inverted phase-contrast microscope, the proliferation, confluence and differentiation of cultured ceils were examined by scanning electron microscope and immunocytochemistry, the basal and stimulated ciliary beat frequency （CBF） of cultured epithelial cells were measured by using high-speed digital microscopic imaging system. Prism 4. 0 software was used to analyze the data. Results （1） Under microscope, the cells on transwell membrane adhered well at 24 h and locked tightly to display with a cobblestone-like appearance; the monolayer cells got confluence to 80% -90% after one-week submersion culture, and thereafter exposed to air-liquid interface. （2） Under scanning electron microscope, the cilia and also the small microvilli could be observed to protrude from the cell＇s surface at ALl day 7 ; the ciliated cells differentiated well and distributed in cluster; goblet cells and nonciliated columnar cells distributed between ciliated cells. （3） Immunocytochemistry of β-tubulin 1V and zona occludens-1 showed a good confluence and differentiation of cilia in cultured epithelial cells at ALl day 14, and the percentage of ciliated epithelial cells was 50% -60 %. （4） The basal CBF of cultured epithelial cells was （ 8.42 ± 1.24）, （ 8.71 ± 1.11 ）, （9. 17 ± 1.11 ）, （ 8.89 ± 0. 91 ）, （ 8.99 ± 0. 91 ） Hz at ALI day 7, 14 , 21, 28, 35, respectively, no significant difference was found among them（F = 1. 451, P 〉0. 05）. （5） At the concentration of 100 μmol/L ATP, an exogenous stimulating agent, significantly increased the CBF of cultured epithelial cells. Conclusions Air-liquid interface cultured human nasal

关 键 词：细胞培养技术 纤毛 上皮细胞 鼻腔 鼻息肉 

分 类 号：R765.25[医药卫生—耳鼻咽喉科]