摘要
目的对正常成年小鼠肺内支气管肺泡干细胞（bronehoalveolar stem cell，BASC）进行克隆培养、鉴定和流式细胞仪分选。方法用胶原酶和分散酶联合消化小鼠肺组织制备肺单细胞悬液，并经免疫磁珠分选Sca-1^＋细胞；胶原蛋白和小鼠Swiss-3T3成纤维细胞系包被培养板；无血清乳腺上皮细胞培养基培养Sca-1^＋细胞；双重免疫荧光染色Clara细胞抗原（CCA）和肺泡表面活性蛋白C（SP-C）鉴定BASC，并用流式细胞仪分选BASC。结果通过胶原酶和分散酶联合消化小鼠肺组织，平均每只成年小鼠可得到有核细胞总数（1．6～1．8）×10^7；经磁珠分选后的Sca-1^＋细胞纯度明显高于未经分选的肺Sca-1^＋细胞[分别为（87．3±5．9）％、（9．6±1．8）％，P〈0．05]；在无血清乳腺上皮细胞培养基中Sca-1^＋细胞在第4天可形成BASC克隆和其他不表达CCA、SP-C的细胞克隆；流式细胞仪检测CD45^-CD31^-Sca-1^＋CD34^＋细胞占肺细胞总数的0．7％～1．1％，根据此分子标记能够分选出高纯度的BASC。结论通过胶原酶和分散酶联合消化，免疫磁珠分选的Sca-1^＋细胞在无血清乳腺上皮细胞培养基中能够形成非纯化的BASC克隆，而流式细胞仪可以分选出高纯度的BASC。
Objective To cultivate, identify and sort bronchoalveolar stem cells （BASC） derived from normal adult mouse lung. Methods After enzymatic digestion of lung tissue with dispase and collagenase in combination, the Sca-1^＋ cells were isolated from the obtained pulmonary cells by magnetic cell sorting. These Sca-1^＋ cells were cultured in dishes coated with collagen and mouse fibroblast cell line Swiss-3T3 under a serum-free culture system for BASC, which were identified by the dual-color immunofluorescent staining clara cell specific antigen （CCA） and surfactant protein C （SP-C）. Finally, these pure BASC were isolated by the flow cytometry. Results One lung of normal adult mouse could yield （ 1.6 - 1.8 ）×10^7 nucleated cells in this enzyme digestion procedure. The percentage of Sca-1^＋ cells we sorted from lung tissue was much higher than the unsorted [ （87.3±5.9）% and （9.6±1.8）%, P 〈0.05]. On the 4th day of culture, the Sca-1^＋ cells grew into BASC and other cell colonies. The CD45^- CD31^- Sca-1 ＋ CD34^＋ cells accounting for （0.7 - 1.1 ） % of the total number of pulmonary cells were isolated by flow cytometry. Conclusion Sca-1^＋ cells derived from adult mice pulmonary cells can grow into BASC in serum-free culture system, and BASC can be purified by flow cytometry. The established platform provides implications for the research of the lung tumor stem cells from human.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2008年第9期775-778,共4页
Journal of Third Military Medical University
基金
国家高技术研究发展计划("863"项目)(2007AA02Z129)
国家自然科学基金(30672076)~~
