脐血CD34^+干细胞体外诱导树突状细胞的表型特征及其影响因素分析  

Analysis of phenotype and influencing factors for cultured dendritic cells originated from cord blood CD34^+ stem cells

作　　者：宋志强[1] 钟华[1] 牛军[1] 陈曙光[1] 邓小蓉[1] 郝飞[1] 

机构地区：[1]第三军医大学西南医院皮肤科,重庆400038

出　　处：《免疫学杂志》2013年第11期933-937,942,共6页Immunological Journal

基　　金：国家自然科学基金资助项目(81072447);重庆市科委自然科学基金计划资助项目(CSTC2010BB5033)

摘　　要：目的建立脐血CD34+干细胞体外诱导树突状细胞(DC)的培养体系,并观察IgE受体和Toll样受体的表达特征及其影响因素。方法无菌条件下常规采集脐血,采用梯度离心试剂和全自动磁性活化细胞分选仪(autoMACS)分离CD34+肝细胞并培养,每4天半量更换含有重组人粒细胞-巨噬细胞集落刺激因子(GM-CSF)和TGF-β的培养基,隔日加入1 ng/ml的人骨髓瘤IgE(myeloma IgE,mIgE)。于培养不同时段收获细胞,利用单克隆抗体和流式细胞术对诱导细胞亚群检测CD1a、CD14、Langerin、FcεRⅠ、FcεRⅡ和TLR的表达。结果体外诱导培养4 d后可观察到典型的树突状细胞,不同质量浓度的TGF-β可影响细胞Langerin、FcεRⅠ和TLR2的表达;Langerin的表达主要限于CD1a+和CD14C+D1a+(DP)细胞亚群,FcεRⅠ和TLR2在不同的细胞亚群均有表达,以CD14+、DP和CD1a+细胞亚群中表达水平较高,不同类型的细胞群不同程度地表达CD1a、Langerin、FcεRⅠ、FcεRⅡ和TLR2。结论利用GM-CSF、TGF-β和mIgE可诱导脐血CD34+干细胞分化为表达FcεRⅠ和TLR2受体的DC,TGF可调节2种受体表达水平。This study designed to establish an in vitro inducing culture system of dendritic cells by using CD34＋ stems cells from cord blood and to study the expression of re1ated cell surface markers. CD34＋ stems cells were iso1ated from cord blood by using gradient centrifugation regent and auto magnetic activated cell sorting system （autoMACS）. The GM-CSF and TGF-β were added into culture medium every four days and mIgE was added every other day. Cultured cells were harvested and analyzed for expression levels of CD1a, CD14, Langerin, FceR Ⅰ, FceR Ⅱand Toll like receptors by flow cytometry. Results showed typical DCs could be observed on day 4. And the expression of Langerin, FceR Ⅰ and TLR2 could be influenced by TGF-β at different concentrations. Langerin cell was mainly expressed on CD1a-expressing subpopu1ations （DP and CD1a）, while FceR Ⅰ and TLR2 could be expressed in different subpopu1ations. DP expressed more FceRI than cells from CD14 or CD1a subpopu1ations did. Results in present study indicated that DC co-expressing TLRs and FceRI could be generated from CD34＋ stems cells of cord blood in the conditioned culture system containing GM-CST, TGF-β and mIgE in vitro. The concentration of TGF-β can influence the expression level of TLR and FcεR Ⅰ.

关 键 词：树突状细胞 炎症性树突状细胞 高亲和力IGE受体 TOLL样受体2 

分 类 号：R392.8[医药卫生—免疫学]