小鼠支气管肺泡干细胞的分离、鉴定与三维培养体系的构建  

Isolation,identification and construction of three-dimensional culture system of mouse bronchoalveolar stem cells

作　　者：王淑艳 陈以文 于雪 刘丹彤 盛春瑞 畅洪昇[2] 李丽娜 WANG Shu-yan;CHEN Yi-wen;YU Xue;LIU Dan-tong;SHENG Chun-rui;CHANG Hong-sheng;LI Li-na(School of Traditional Chinese Medicine,Beijing University of Chinese Medicine,Beijing 100029,China;School of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing 100029,China)

机构地区：[1]北京中医药大学中医学院,100029 [2]北京中医药大学中药学院,100029

出　　处：《蚌埠医学院学报》2020年第1期1-4,共4页Journal of Bengbu Medical College

基　　金：教育部留学回国人员科研启动基金教外司留(2014)1685号。

摘　　要：目的:探讨C57BL6小鼠支气管肺泡干细胞(BASCSs)体外分离、鉴定及其三维培养体系的构建方法,为进一步研究BASCSs的相关特性奠定实验基础。方法:选用C57BL6成年雄鼠,完整分离肺组织,1×PBS灌洗后,经支气管分次灌注Elastase酶进行消化,以锐器切碎肺组织,加入DNA酶使细胞更好地分散开。用75μm细胞筛去除未消化的组织块,所得细胞悬液以荧光标记的CD31、CD34、CD45、SCA-1、EpCAM抗体染色,流式细胞仪分选CD31-CD34-CD45-SCA-1+EpCAM+细胞,细胞与Mlg2908细胞(小鼠肺成纤维细胞株)按(1×103)∶(1×106)的比例混合,以Matrigel为基质构建三维培养体系。结果:通过Elastase酶消化肺组织,每只成年小鼠可得到有核细胞总数(1.6~1.8)×107个,流式细胞技术结果显示,CD34^-CD45^-SCA-1^+EpCAM+细胞约占EpCAM+细胞的22%;将支气管肺泡干细胞、Mlg2908细胞和Matrigel混合培养,4 d后可见克隆形成。随着培养时间延长,克隆数量逐渐减少。8 d后大部分克隆直径达50μm,形态出现分化,克隆可维持至15 d。结论:建立了一种简单、高效的分离、鉴定及三维培养小鼠BASCSs的方法。Objective:To explore the method of isolation,identification and three-dimensional culture system of bronchioalveolar stem cells(BASCSs)from C57BL6 mouse in vitro,so as to lay an experimental foundation for further study of related characteristics of BASCs.Methods:The lung tissue of adult male C57BL6 mice was completely isolated,lavaged by 1×PBS,digested by Elastase solution perfused through the trachea.The lung tissue was cut up with a sharp instrument,the cells were better dispersed using DNA enzymes,and the undigested tissue was removed with 75μm sieve.The cells suspensions were stained using the fluorescent labeled CD31,CD34,CD45,SCA-1 and EpCAM antibody.The CD31-CD34-CD45-SCA-1+EpCAM+cells were sorted out using flow cytometry,and mixed with Mlg2908 cells(mouse lung fibroblast cell line)at the ratio of(1×103)∶(1×106)to construct a three-dimensional culture system based on the Matrigel as matrix.Results:The lung tissue was obstained by Elastase digesting,the 1.6×107 to 1.8×107 nucleated cells were obtained from a mouse.The results of flow cytometry showed that the CD34^-CD45^-SCA-1^+EpCAM+cells accounted for about 22%of EpCAM+cells.BASCSs,Mlg2908 cells and Matrigel were mixed and cultured.After 4 d,the clone was observed.With the extension of culture time,the number of clones decreased gradually.After 8 days,most of the clones were 50μm in diameter,differentiated in morphology,and could sustain for 14 d.Conclusions:A simple and efficient method for isolation,identification and three-dimensional culture of mouse BASCSs is successfully established.

关 键 词：支气管肺泡干细胞 细胞分离 细胞三维培养 小鼠 

分 类 号：Q813[生物学—生物工程]