抗CD40抗体联合诱导人脐血CD34^+造血干细胞向DC2分化的作用

Effects of CD40 signal on induction of DC2 differentiation from CD34^+ hemopoietic cell of human cord blood in vitro

目的探讨从脐血CD34+造血细胞诱导DC2的方法及CD40分子在DC2分化诱导中的作用。方法运用磁珠从脐血中分离出CD34+造血干细胞,以rhIL3(10ngmL)、rhFlt3L(100ngmL)和rhGMCSF(100ngmL)诱导其向DC2分化,采用流式细胞仪分析DC2表型,并观察抗人CD40单克隆抗体诱导DC2分化成熟的作用。结果人脐血CD34+造血细胞在rhIL3、rhFlt3L和rhGMCSF联合诱导培养12d,获得具有DC2样(淋巴样DC)形态的细胞,然后分别加入rhIL3+抗人CD40mAb(Ⅰ组)或rhIL3+TNFα(Ⅱ组)诱导DC2的分化成熟,流式细胞仪分析细胞表型,发现Ⅰ组和Ⅱ组Lineage-(CD3、CD14、CD16、CD19、CD56)CD123+细胞的HLADR、CD83、CD86、CD80表达率分别为88.78%、37.38%、32.83%和99.08%;78.87%、32.29%、29.57%和98.86%。结论体外联合多种细胞因子从CD34+造血细胞成功地诱导出富集DC2表型的细胞,抗人CD40mAb可以促进DC2的分化成熟。

Objective To develop a method of inducing DC2 from human cord blood CD34^＋ hemopoietic cells and evaluate the effect of CD40 signal on differentiation and induction of DC2. Methods CD34^＋ hemopoietic cells were isolated from cord blood of healthy volunteers withCD34-FITC and anti-FilE labeled magnetic microbeads, then cultured with rhIL-3 （10 ng/mL）, rhFh-3L （100 ng/mL）, and rhGM-CSF （ 100 ng/mL）. The surface molecules of DC2 were identified by FCM and the role of anti-CD40 mAb in differentiation and maturation of DC2 was analyzed by cytokines assay. Results CD34^＋ hemopoietic cells of human cord blood were cultured with rhIL-3, rhFlt-3L, and rhGM-CSF for 12 days, and then rhIL-3 ＋ anti-human CD40mAb （group Ⅰ ） or rhIL-3 ＋ TNF-α （groupⅡ） were added respectively to induce their differentiation and maturation. The expression rates of surface molecules HLA-DR, CD83, CD86, and CD80 on Lineage^- CD123^＋ DC2 induced by group Ⅰ were 88.78%, 37.38%, 32.83%, and 99.08%, respectively; otherwise the same surface molecules on Lineage^- CD123^＋ DC2 induced by groupⅡ were 78.87%, 32.29%, 29.57%, and 98.86%, respectively. Conclusion DC2 can be induced from cord blood CD34^＋ hemepoietic cells with cytokines cocktail in vito. The agonist anti-human CD40mAb can enhance the differentiation and maturation of DC2.