明胶法富集外周血单核细胞并刺激其成熟为树突状细胞

Purification of human peripheral blood monocytes on gelatin-coated surface and stimulation into dendritic cells

背景:如何简易高效分离纯化人外周血单核细胞并刺激成熟为树突状细胞,未见标准化操作流程。目的:观察明胶法分离外周血单核细胞的效率以及将分离出的单核细胞刺激成熟为树突状细胞的表型特征,并与普通塑料黏附法对比。方法:使用人淋巴细胞分离液分离人外周血得到单个核细胞,根据培养瓶是否进行明胶包被分为明胶包被组和普通塑料组。均分单个核细胞,按组别分离获得单核细胞并诱导刺激成熟为树突状细胞。计数各组所得单核细胞数,使用流式细胞仪检测2组单核细胞的CD14阳性率、T、B淋巴细胞污染率、树突状细胞非成熟期和成熟期CD1a,CD83的表达情况,锥虫蓝拒染法计算细胞活率,观察对比2组血小板污染情况。结果与结论:明胶包被组单核细胞数及CD14阳性率显著高于普通塑料组(P<0.05),普通塑料组淋巴细胞污染率显著高于明胶包被组(P<0.05)。2组细胞活率及树突状细胞表型差异无显著性意义(P>0.05)。明胶包被组血小板污染率低于普通塑料组。提示明胶法可以简单高效分离出单核细胞并成功刺激成熟为树突状细胞。

BACKGROUND：There have been no standard procedures regarding how to simply and efficiently separate and purify human peripheral blood monocytes and stimulate them into dendritic cells.OBJECTIVE：To investigate the efficacy of purification of human peripheral blood monocytes on gelatin-coated surfaces,analyze the phenotype of dendritic cells generated by these monocytes,and make a comparison with conventional plastic adhesion method.METHODS：Human peripheral blood mononuclear cells were harvested by Ficoll-Hypaque gradient centrifugation.Gelatin group and common plastic group were designated according to coating flasks with or without gelatin.Monocytes were harvested from each group and then were stimulated into dendritic cells.The number of monoctyes,CD14 positive rate of monocytes,contaminated T,B lymphocytes,the expression of CD1a,CD83 on immature and mature dendritic cells were determined.Cell viability was determined by trypan blue staining.Platelet contamination was compared between gelatin and common plastic groups.RESULTS AND CONCLUSION：The number of monocytes and CD14 positive rate were significantly higher in the gelatin group than in the common plastic group（P 0.05）.Contaminated T,B lymphocytes were significantly more in the common plastic group than in the gelatin group（P 0.05）.There were no significant differences in cell viability and dendritic cell phenotype between gelatin group and common plastic group（P 0.05）.Platelet contamination rate was significantly lower in the gelatin group than in the common plastic group.Gelatin-coated surfaces can effectively and simply isolate monocytes and successfully stimulate them into mature dendritic cells.