未成熟树突状细胞快速分离与体外诱导培养及其鉴定研究

摘要

目的:以人外周血来源的单核细胞为前体细胞,建立体外快速分离和诱导培养未成熟树突状细胞(Immature dendritic cell,iDC)的方法.方法:采用Ficoll密度梯度离心方法和MACS磁珠分选系统,收集高纯度CD14+单核细胞;用rhGM-CSF、rhIL-4联合诱导培养CD14+单核细胞,第4天获得未成熟树突状细胞(iDC),应用流式细胞术检测细胞表面标记(CCR5)和抗原吞噬能力,普通光学显微镜、扫描电镜和透射电镜观察细胞表面和内部结构特征.结果:CD14免疫磁珠技术获得CD14+单核细胞纯度达94%以上,诱导分化第4天的iDC具有CCR5特征性标志和吞噬能力,普通光学显微镜及电镜观察到iDC出现树突状细胞典型特征.结论:体外快速诱导培养是获得大量具有典型特征的iDC的有效方法,并可应用于进一步实验研究.%Objective:To establish a fast method for the generation of immature dendritic cells(DCs) from human peripheral blood mononuclear cells(hPBMCs) in vitro.Methods: High purity human CD14+ monocytes were collected using density Ficoll gradient centrifugation and MACS beads sorting system.iDCs(Immature DC) were induced after cultured with rhGM-CSF and rhIL-4 on the fourth day.Fluorescence activated cell sorting(FACS) was used to identify cell surface markers(CCR5) and capabilities of antigen uptake of iDCs on the fourth day.Ordinary optical microscope,scanning electron microscopy and transmission electron microscopy were used to observe surface and internal structure of iDCs on the fourth day of culture conditions.Results: FACS result shows that the purity of CD14+ monocytes collected from hPBMCs were more than 94%.The antigen uptake capability and CD195 of iDCs was detected on the fourth day of cultured conditions.Typical surface and internal structure characteristics of iDCs were observed.Conclusion: Rapid induction culture is an effective method for obtaining a large number of iDC with typical characteristics in vitro,and can be used for further experimental study.