人PBMC和脐血CD34~+细胞诱导DC2的实验方法研究

目的:探讨从人外周血单个核细胞(PBMC)和脐血CD34+造血干细胞(HSC)诱导DC2方法。方法:分别用rhIL-3(10ng/ml)+rhG-CSF(100ng/ml)和rhFlt-3L(100ng/ml)+rhIL-3(10ng/ml)+rhG-CSF(100ng/ml)诱导人PBMC和脐血CD34+HSC向DC2分化,并采用流式细胞仪分析细胞的表型,同时观察rhTNF-α和人CD40单抗诱导DC2分化成熟的作用。结果:rhG-CSFh+rhIL-3未能较好地从PBMC中诱导出DC2,但是从富集的脐血CD34+HSC用rhFlt-3L+rhIL-3+rhG-CSF诱导,可以获得相对较多的DC2,并有较高的扩增效率,但离临床研究和应用还有一定距离,因此DC2的诱导方法仍需进一步优化。人CD40单抗和rhTNF-α一样,能有效地诱导DC2分化成熟。结论:体外联合多种细胞因子从CD34+HSC成功地诱导出DC2,抗人CD40单抗有利于DC2的活化成熟。

Establish a method for Inducing DC2 via CD34+ hematopoietic stem cells from Human cord blood or PBMC

Objective:To investigate the conditions of inducing DC2 via human peripheral blood mononuclear cells (PBMC) or CD34+ hematopoietic stem cells (HSC) enriched from human cord blood and evaluate the effect of anti-CD40 McAb in inducing DC2 maturation. Methods:PBMC was isolated from blood and was CD34+ HSC enriched from cord blood of healthy volunteers respectively. The pre-DC from PBMC or CD34+ HSC were generated with rhIL-3(10ng/ml), rhG-CSF(100ng/ml) or rhIL-3(10ng/ml), rhG-CSF(100ng/ml) and rhFlt-3L(100ng/ml), and then was the membrane surface mark of DC2 with FCM identified. Results:CD123+DC2 were successfully induced from CD34+ cord blood HSC with rhFlt-3L,rhIL-3 and rhG-CSF, but were unsuccessfully induced from PBMC with rhIL-3 and rhG-CSF in vitro. Furthermore, DC2 were induced to maturation by anti-human CD40 McAb.Conclusions:DC2 can be induced from CD34+ cord blood HSC with rhFlt-3L,rhIL-3 and rhG-CSF and be induced to maturation by anti-human CD40 McAb in vitro.