利用人气道基底细胞体外构建类气管球  

Preparation of bronchospheres generated from human airway basal cells

作　　者：张文平 刘姿 贾建超 魏立 张晓菊 ZHANG Wenping;LIU Zi;JIA Jianchao;WEI Li;ZHANG Xiaoju(Department of Respiratory and Critical Care Medicine,Henan Provincial People′s Hospital(Zhengzhou University People′s Hospital,Henan University People′s Hospital),Zhengzhou 450003;Department of Thoracic Surgery,Henan Provincial People′s Hospital(Zhengzhou University People′s Hospital,Henan University People′s Hospital),Zhengzhou 450003)

机构地区：[1]河南省人民医院(郑州大学人民医院,河南大学人民医院)呼吸与危重症医学科,郑州450003 [2]河南省人民医院(郑州大学人民医院,河南大学人民医院)胸外科,郑州450003

出　　处：《郑州大学学报（医学版）》2022年第2期228-232,共5页Journal of Zhengzhou University(Medical Sciences)

基　　金：河南省基础与前沿技术研究项目(162300410109);河南省卫健委医学科技攻关计划项目(201702227)。

摘　　要：目的:利用人气道基底细胞体外培养类气管球。方法:利用河南省人民医院肺移植术中弃用的供肺分离并培养人气道基底细胞、肺成纤维细胞。将气道基底细胞和肺成纤维细胞1∶1混合,重悬于基质胶,用EpiX Base培养基培养,以单独培养的基底细胞或肺成纤维细胞为对照。观察是否形成类气管球及不同时间点类气管球的形态,并采用免疫荧光染色明确成熟类气管球的细胞成分。将气道基底细胞老年株或青年株分别和人肺成纤维细胞老年株或青年株联合培养,观察形成的类气管球的差别。结果:气道基底细胞单独培养或与肺成纤维细胞联合培养均可见类气管球形成,肺成纤维细胞单独培养无类气管球形成。类气管球在镜下呈球形,随着培养时间延长球体增大,14~21 d达峰值。免疫荧光染色可见KRT5+P63+基底细胞分布于类气管球球体表面,FOXJ1+纤毛细胞和MUC+分泌细胞分布在管腔内部。培养第20天,与基底细胞青年株相比,基底细胞老年株与成纤维细胞联合培养所形成的类气管球直径更大,数量更多。结论:人气道基底细胞体外培养可形成类气管球,人肺成纤维细胞和气道基底细胞联合培养可促进类气管球的形成。来自老年宿主的基底细胞体外培养所形成的类气管球的直径大于青年宿主且数量更多。Aim:To generate bronchospheres in vitro using human airway basal cells.Methods:The abandoned donor lungs from the Lung Transplant Center of Henan Provincial People′s Hospital were used to isolate human airway basal cells and lung fibroblasts.The airway basal cells and lung fibroblasts were mixed(1∶1),resuspended in matrigel,and cultured in EpiX Base medium.For the controls,airway basal cells or lung fibroblasts were cultured separately.Cells in the matrigel were checked every day to observe whether bronchospheres formed and the morphology of the bronchospheres at different time points.The immunofluorescence staining was performed to confirm the cellular components of mature bronchospheres.Simultaneously,airway basal cells from old and young healthy hosts were co-cultured with old or young lung fibroblasts to observe the differences in bronchospheres formation.Results:Bronchospheres were observed both in airway basal cells/lung fibroblasts mix group and airway basal cells alone group.There was no bronchosphere formation when lung fibroblasts were cultured alone.The bronchosphere was spherical under the microscope,and the diameter of the spheroid gradually increased with the extension of the culture time,reaching a peak at 14-21 days.Immunofluorescence staining showed that basal cells(KRT5+P63+)distributed on the surface of the bronchospheres,and ciliated cells(FOXJ1+)and secretory cells(MUC+)were distributed inside the lumen.Old airway basal cells and lung fibroblasts mixture culture formed more bronchospheres than young basal cells and lung fibroblasts,and the diameter of bronchospheres was larger.Conclusion:Human airway basal cells cultured in matrigel in vitro could form bronchospheres.The mixed culture of lung fibroblasts and airway basal cells can promote the formation of bronchospheres.Basal cells from old hosts have higher efficiency in forming bronchospheres than those from young hosts,with more bronchospheres and larger diameter.

关 键 词：人气道基底细胞 人肺成纤维细胞 类气管球 体外类肺器官 

分 类 号：R655.3[医药卫生—外科学]