小鼠肺间质树突状细胞的分离、纯化与鉴定

The separation,purification and identification of mouse interstitial dendritic cells in lung

目的：建立肺间质树突状细胞（DCs）的分离与纯化方法,为肺间质DCs的相关研究提供实验基础。方法：雄性BALB/c小鼠肺组织经Ⅰ型胶原酶消化、密度梯度离心、CD11c＋免疫磁珠分选纯化DCs,流式细胞术鉴定分选DCs纯度,倒置相差显微镜观察孵育细胞生长状态,扫描电镜和透射电镜观察DCs超微形态,流式细胞术检测肺间质DCs的CD11c、CD11b、CD86和I-Ab表型。结果：分选获得的肺间质DCs经鉴定,纯度为92.59%±5.62%,在RPMI1640培养基中生长状态良好,少数细胞形成小细胞集落。DCs超微形态观察显示细胞表面多见长1~2μm密集的树枝状突起,细胞器不发达,细胞核形不规则。90%以上肺间质DCs为未成熟状态或前体DCs,低表达成熟标志物I-Ab和CD86,并具有异质性,近40%起源于髓系细胞分化。结论：密度梯度离心和免疫磁珠分选是分离、纯化肺间质DCs的理想方法,获得的DCs纯度高、活性好、免疫表型稳定,但操作步骤比较复杂,技术要求高。

Objective：To establish the methods to separate and purify pulmonary interstitial dendritic cells（DCs） of mice in order to provide experimental base for the related study on them.Methods：Pulmonary interstitial DCs of male BALB/c mice were separated by lung tissue digesting and density gradient centrifugation,and were purified by CD11c＋microbeads.Growth state of DCs was observed by phase contrast microscope.Ultrastructure of DCs was observed by scanning and transmission electron microscope.Immunological phenotype of DCs was analyzed with CD11c,CD11b,CD86 and I-Ab by flow cytometry.Results：The purity of pulmonary DCs acquired by separation with immunomagnetic beads was 92.59%±5.62%,and they grew in good state in culture media.DCs had coarctate dendritc process of 1-2 μm in length in the surface,incomplete organelle in cytoplasm and irregular nuclei.Over 90% of pulmonary DCs were immature or pre-DCs expressing low level of molecular markers I-Ab and CD86,and they were heterogeneous about 40% of them originating from myeloid cells.Conclusion：Pulmonary DCs acquired by density gradient centrifugation and immunomagnetic beads had high purity and stable immunological phenotype.They were relatively practical methods to separate and purify pulmonary DCs in spite of complex procedure and technique.