人脐血造血干细胞体外诱导朗格汉斯细胞的研究

In vitro induction of Langerhans cells from human umbilical cord blood hematopoietic stem cells

目的采用人脐血CD34+细胞,经不同细胞因子组合诱导方案体外诱导培养朗格汉斯细胞(CD1a+细胞)并观察其产量及相关表型特点。方法联合应用人淋巴细胞分离试剂和CD34+磁珠分离和筛选人脐血CD34+细胞,以5种不同的细胞因子组合诱导方案,对分选出的CD34+细胞进行体外诱导培养,观察其生长形态变化、集落的形成,并对诱导培养第12天的CD34+细胞进行CD1a、HLA-DR等LC表型分子,以及CD40、CD80等共刺激分子表型检测。筛选出最佳的朗格汉斯细胞(Langerhans cells,LC)体外诱导方案。结果流式细胞术分析5种细胞因子方案体外诱导培养LC的结果显示,方案IV、V诱导培养12 d的CD1a+细胞百分率和CD1a+细胞数量均明显多于方案Ⅰ、Ⅱ和Ⅲ;方案Ⅳ诱导培养12 d的HLA-DR+细胞百分率显著高于方案Ⅴ。结论利用细胞因子组合方案IV体外诱导磁珠分选的CD34+细胞,可以获得高产量的LC细胞,为研究其功能和发生机制提供足够的细胞。

The study was designed to screen the protocols of various combinations of cytokines for inducing Langerhans cells from human umbilical cord blood CD34＋ cells, and analyze the expression of related cell surfacemarkers. The mononuclear cells （MNCs） were separated from human umbilical cord blood by human lymphocyte separation medium. The CD34＋ cells were then further purified from MNCs by direct CD34 immunomagnetic beadsand magnetic activated cell sorting （MACS）. Subsequently, the CD34 ＋ cells were cultured with five various combinations of cytokines in vitro for 12 day for Langerhans cells induction. Langerhans cells with CDla＋ phenotypewere identified by flow eytometry analysis, and the protocol with high yield of CDIa＋ Langerhans cells was optimized. The other cell surface markers, such as HLA-DR, CD40 and CD80, were also measured. As a result,after 12 days culture with five combinations of cytokines, CDla＋ cells induced from CD34＋ cells by protocols （ Ⅰ , Ⅱ, Ⅲ,Ⅳ, and Ⅴ） were （31.81±1.83）%, （24.87±0.67）%, （26.24±4.67）%, （51.61±1.65）% and （52.94±1.26）%, respectively.The protocols of Ⅳ and Ⅴ induced more CDla＋ cells than the other three protocols did. Furthermore, after 12 days cultivation, the protocol Ⅳ induced more CDla＋HLA-DR＋ cells than the protocol V did. So four cytokines＇combination in protocol Ⅳ can induce more Langerhans cells （CDla＋ ceils） and HLA-DR＋ cells from CD34＋ cells after 12 days＇ culture in vitro, which should be enough for the study of Langerhans cells＇ functions and mechanisms.