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

王淑艳; 陈以文; 于雪; 刘丹彤; 盛春瑞; 畅洪昇; 李丽娜

doi:10.13898/j.cnki.issn.1000-2200.2020.01.001

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

摘要

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75 μm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，4d后可见克隆形成。随着培养时间延长，克隆数量逐渐减少。8d后大部分克隆直径达50 μm，形态出现分化，克隆可维持至15d。
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。

Abstract:
ObjectiveTo 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.
MethodsThe 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 75 μm 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.
ResultsThe 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 50 μm in diameter, differentiated in morphology, and could sustain for 14 d.
ConclusionsA simple and efficient method for isolation, identification and three-dimensional culture of mouse BASCSs is successfully established.

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