气道和肺泡部位成纤维细胞的原代分离和培养  

Isolation and culture of primary fibroblasts derived from airway and alveolar tissues

作　　者：曾莹莹 胡蔚萍 左依慧 张静[1] ZENG Yingying;HU Weiping;ZUO Yihui;ZHANG Jing(Department of Pulmonary and Critical Care Medicine,Zhongshan Hospital,Fudan University,Shanghai 200032,China)

机构地区：[1]复旦大学附属中山医院呼吸与危重症医学科,上海200032

出　　处：《上海医药》2021年第3期68-72,共5页Shanghai Medical & Pharmaceutical Journal

基　　金：国家自然科学基金资助项目(81470231,81670030)。

摘　　要：目的:建立小鼠气道和肺泡部位原代成纤维细胞的分离、培养方法,为研究肺内成纤维细胞的区域性差异提供技术平台。方法:将小鼠双肺取出并固定后,在体视显微镜视野内分别分离气道和肺泡组织块,并应用组织块培养法分别进行细胞的体外原代和传代培养。通过细胞形态观察以及细胞表面标志物的免疫荧光检测对培养的成纤维细胞进行鉴定。此外,采用阿尔玛蓝法分别检测气道和肺泡部位成纤维细胞的增殖情况并进行比较。结果:12 h内可见分离的原代细胞贴壁生长,7 d可进行传代。在倒置显微镜下观察,细胞呈长梭状,胞体丰满,胞质均匀,符合成纤维细胞的特点,并且细胞在5代以内可保持良好的生长状态和稳定的性状。免疫荧光检测结果显示,抗波形蛋白染色和抗α-平滑肌肌动蛋白染色呈阳性,抗细胞角蛋白染色、抗血管性假血友病因子染色、抗结蛋白染色均呈阴性,由此表明该分离培养的细胞为成纤维细胞,且纯度高达99%。此外,气道部位的成纤维细胞增殖能力显著高于肺泡部位。结论:应用显微解剖加组织块法可成功分离培养小鼠气道和肺泡部位成纤维细胞,并且使细胞在5代以内生长状态良好,从而为探索小气道壁和肺泡成纤维细胞功能差异提供必要的细胞工具。Objective:To establish mice fibroblasts of airway and alveolar tissues isolation method for primary culture and research use and to provide the platform for the research of regional differences between fibroblasts from the airway and alveolar tissues.Methods:The 6-8 month old mice were sacrificed and the lung were got totally together with trachea.Then the airway tissues and the alveolar tissues were isolated under the stereomicroscope respectively.Primary fibroblasts were cultured by tissue block culture method and were identified by cell morphology and immunofluorescence staining.The proliferative ability of fibroblasts derived from airway and alveolar tissues was analyzed by Alamar blue assay.Results:The fibroblasts derived from airway and alveolar tissues were generally elongated and spindle shaped and were in line with the classic fibroblasts form.Moreover,the primary fibroblasts maintained the steady growing condition within the 5 passages.Further analysis using immunofluorescence revealed that the fibroblasts markers vimentin andα-SMA were positive,cytokeratin staining,desmin staining,and von Willebrand factor staining were negative,which showed that the primary fibroblasts were fibroblasts.In addition,the proliferative ability of fibroblasts derived from airway tissues was significantly higher than that from alveolar tissues.Conclusion:Primary fibroblasts derived from airway and alveolar tissues could maintain good condition within passage 5.And it provided a good cell tool to investigate the functional difference between this kind of fibroblasts.

关 键 词：成纤维细胞 气道组织 肺泡 小鼠 显微分离 原代培养 

分 类 号：Q813.11[生物学—生物工程]