小鼠肺间质树突状细胞的分离_纯化与鉴定_王宏伟

¹本文为国家自然科学基金资助项目(81000848)º军事医学科学院微生物流行病研究所,北京100071

作者简介:王宏伟(1976年-),男,博士,副主任医师,主要从事血液

病理学及免疫病理学研究,E -mail:whw6626@s ;

通讯作者及指导教师:陆江阳(1957年-),男,硕士,主任医师,博士

生导师,主要从事创伤与危重病免疫病理学研究,E -mail:luj y@ 。

doi:10.3969/j.issn.1000-484X.2011.09.012

#免疫学技术与方法#

小鼠肺间质树突状细胞的分离、纯化与鉴定¹

王宏伟 陆江阳 田 光º 刘 茜 赵 敏 杨 毅 康佳蕊(解放军总医院第一附属(304)医院病理科,北京100048)

中国图书分类号 R36417 文献标识码 A 文章编号 1000-484X (2011)09-0814-05

[摘 要] 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Ñ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 -A b 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1~2L m 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 -A b 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。

[关键词] 肺;树突状细胞;免疫磁珠;CD11c

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

WANG Hong -Wei,LU Jiang -Yang,TIAN Guang ,LIU Qian,Z HAO Min,YANG Yi,KANG Jia -Rui.Department o f Pathology ,the First Affiliated Hos pital o f General Hospital o f P LA ,Beijing 100048,China

[Abstract] Objective:To establish the methods to separate and purify pulmonary i nterstitial dendri tic cells(DCs)of mice in order to provide experimental base for the related study on them.Methods:Pul monary in terstitial DCs of male B ALB/c mice were separated by lung tis -sue diges ting and density gradient centrifugation,and were puri fied 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 ana -lyzed wi th CD11c,CD11b,CD86and I -A b by flow cytometry.Results:The purity of pul monary DCs acquired by separation wi th im munomagnet -ic beads was 92.59%?5.62%,and they grew in good state in cul ture media.DCs had coarctate dendritc process of 1-2L m 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 -A b and CD86,and they were heterogeneous about 40%of them originating from myeloid cells.Conclusion:Pulmonary DCs acquired by densi ty gradient centrifugation and immunomagnetic beads had high purity and stable immunolog ical phenotype.They were relatively practical methods to separate and purify pul monary DCs in spite of complex procedure and techniq ue.

[Key w ords] Lung;Dendritic cells;Immunomagnetic beads;CD11c

树突状细胞(Dendritic cells,DCs)是一类功能强大的专职抗原提呈细胞,在机体免疫应答和免疫调

控中起关键作用。DCs 主要分布在免疫器官中,而分布于肺、肝、肾等非淋巴器官中的DC s 被称作间质DCs [1]

。近年来的研究表明,肺间质DCs 与肺感染、

哮喘、移植及创伤性免疫紊乱性疾病的发病机制密切相关,探讨DCs 的免疫功能对阐明上述疾病的机制和防治具有重要意义。由于肺间质DCs 数量稀少,分离、纯化难度大,限制了对其的深入研究。本文探讨小鼠肺间质DCs 的分离、纯化和鉴定方法,为相关研究提供实验基础。

#814#中国免疫学杂志2011年第27卷