一种原代肺上皮细胞体外培养方法

One Method of Primary Alveolar Epithelial Cells in vitro

目的:建立一种操作简便、重复性好的体外培养BALB/c小鼠原代肺上皮细胞(AEC)的方法,探究不同发育阶段小鼠AEC中柯萨奇病毒和腺病毒受体(CAR)表达量的变化,及其对小鼠原代AEC贴壁的作用。方法:手术获取小鼠肺组织,机械法剪碎肺组织,PBS缓冲液清洗肺组织块数次去血,联合使用链霉蛋白酶和胶原酶Ⅰ消化、分离肺组织块获得单细胞悬液,差速离心逐步清除其他种类的细胞,达到纯化AEC的作用。细胞在Ⅰ型鼠尾胶原蛋白包被过的细胞板中培养,光学显微镜下观察不同发育阶段AEC贴壁、生长状态;免疫荧光法鉴定AEC,检测AEC中CAR的表达量。结果:体外获得不同发育阶段BALB/c小鼠的原代AEC;胎鼠、幼鼠AEC贴壁并开始增殖所需时间较成体鼠短;胎鼠及幼鼠AEC中CAR的表达量明显较成体鼠高。结论:建立了稳定可重复的分离、纯化、体外培养小鼠原代AEC的方法,证明了AEC中的CAR可以促进原代AEC贴壁,为完善原代细胞培养方法提供了科学依据。

Objective： To Establish a simple, reproducible BALB/c mouse primary alveolar epithelial cells （AEC） method in vitro. To explore the change of coxsackievirus and adenovirus receptor（CAR） expression in mouse lung epithelial cells of different developmental stages and its impact on the role of primary AEC adherent. Methods： To isolate lung epithelial ceils and obtain a single cell suspension, we washed up the blood of lung tissues which was obtained through surgery and then have been minced mechanically with PBS buffer for several times. To purify lung epithelial cells, pronase and collagenase I have been used to digest connective tissues. Then, miscellaneous cells has been removed gradually by differential centrifugation. Cells were cuhured in the cell plates coated by rat tail collagen I. Adhesion and growth of AEC at different developmental stages were observed by optical microscope. Primary AEC were identified by immunofluorescence, so as the expression of CAR in AEC. Results： Different developmental stages of BALB/c mouse primary AEC were obtained successfully in vitro. Here we demonstrated that AEC of fetal mouse and young mouse required shorter time than adult rats to adherent and proliferate, and have a significantly higher CAR expression. Conclusion： Here we described a stable separation, purification method of mouse primary AEC in vitro. Our results suggest that CAR can promote primary AEC adherent, providing a scientific basis for the improvement in the method of primary cell cuhure.