气液界面培养小鼠气管上皮细胞模型构建  

Culture and differentiation of mouse tracheal epithelial cells at air-liquid interface

作　　者：冀晓丽 胡玥[1] 盛云华[1] 唐黎明[1] JI Xiao-li;HU Yue;SHENG Yun-huan;TANG Li-ming(Dept of Pharmacology and Toxicology/ Center for Drug Safety Evaluation, Shanghai Institute for Food and Drug Control, NMPA Key Lab for Quality Analysis of Chemical Drug Preparations, Shanghai 201203, China)

机构地区：[1]上海市食品药品检验所药理毒理室/药物安全评价中心,国家药品监督管理局化学药品制剂质量分析重点实验室,上海201203

出　　处：《中国药理学通报》2021年第2期282-288,共7页Chinese Pharmacological Bulletin

基　　金：上海市科学技术委员会“科技创新行动计划”(No 19DZ2202700)。

摘　　要：目的构建小鼠气管上皮细胞原代培养模型,应用于外源化合物等的吸入暴露毒性评价。方法通过优化培养基配比成份,分离小鼠气管,蛋白酶低温消化得到上皮细胞,接种到预先包被胶原的Transwell半透膜中,通过细胞跨上皮电阻值与紧密连接蛋白的检测,评估细胞间紧密连接与细胞极化的形式。转换气液界面培养,标记纤毛蛋白,拍摄纤毛形态特征。结果MTEC分离分化培养后,MUC5AC蛋白,乙酰化α-微管蛋白(α-tubulin),β-微管蛋白-Ⅳ(β-tubulin-Ⅳ),闭合小环蛋白(ZO-1)的表达及紧密连接的形成与假复层上皮的形成均与小鼠体内气管组织结构类似,扫描电镜观察小鼠气管上皮细胞表面纤毛形态。结论本研究构建的小鼠气管上皮细胞培养方案,体外培养成功获得具有类似组织结构和生理功能的假复层纤毛柱状上皮,为气道上皮细胞的分离、分化和检测提供了稳定、可靠地方法。Aim Air-liquid interface(ALI)cultures of mouse tracheal epithelial cells(MTEC)are a well-established model to study airway epithelial cells.MTEC provides a powerful approach for the evaluation of the inhalation toxicological in vitro.Methods C57BL/6 mouse tracheal-bronchial epithelial cells were obtained by digestion with protease in cold temperature overnight,and the digestion time was optimized to ensure the quantity and viability of the obtained cells.The cells were cultured into collagen coated Transwell inserts.Proliferating phase and air-liquid interface culture were promoted with different culture media.The expression of tight junction protein and cell trans-epithelial electrical resistance(TEER)were used to evaluate the formation of tight junction between cells and the analysis of cell polarity.The cilia structure was confirmed by electron microscopy and immunofluorescence.Results Highly purified and viable primary airway epithelial cells could be harvested and subcultured by our methods,including morphology and immunocytochemistry staining confirmed the expression of MUC5AC,α-tubulin,β-tubulin-Ⅳand ZO-1.The development of tight-junctions and epithelium were similar with pseudostratified ciliated columnar epithelium morphology.Conclusions A comprehensive protocol for ALI culture was established,reproducing the characteristic pseudostratified ciliated columnar epithelium morphology and physiological functions in vitro.The MTEC protocol provides a stable and reliable method for the isolation,mucociliary differentiation and reproducing.

关 键 词：气管 气道上皮 气液界面 纤毛细胞 粘液细胞 

分 类 号：R-332[医药卫生—人体解剖和组织胚胎学] R322.33[医药卫生—基础医学]