论文部分内容阅读
目的建立一种稳定的体外分离培养和鉴定人脐血内皮祖细胞(endothelial progenitor cells,EPCs)的方法,并探讨利于EPCs增殖的最佳接种浓度。方法采用密度梯度离心法分离得到单个核细胞,制成1×106/ml、1.5×106/ml、2×106/ml、2.5×106/ml、3×106/ml、3.5×106/ml、4×106/ml7个密度接种于铺被人纤维连接蛋白的培养瓶,比较各接种密度的贴壁细胞数和细胞集落形成情况,并采用激光共聚焦显微镜、流式细胞仪和免疫荧光鉴定EPCs。结果不同接种密度得到的贴壁细胞数和细胞集落数有差别,在2.5×106/ml密度接种时得到的最佳贴壁细胞数和细胞集落数,与其他密度比较差异有统计学意义(P<0.05);双染色阳性细胞为正在分化EPCs;流式细胞仪检测内皮祖细胞对CD34、AC133、VEGFR2的阳性表达率分别为(1.67±0.35)%、(12.63±9.70)%、(66.81±6.63)%;免疫荧光检测内皮祖细胞一致性表达CD34、AC133、VEGFR2;黏附能力和迁移能力检测结果分别为(33.7±1.6)、(29.2±1.2)。结论采用Ficoll密度梯度离心法结合贴壁筛选法可以较好地分离人脐血EPCs,以2.5×106/ml的密度接种培养可获得良好的细胞分化和增殖。
OBJECTIVE: To establish a stable method for the isolation and identification of endothelial progenitor cells (EPCs) from human umbilical cord blood in vitro and to explore the optimal inoculation concentration for EPCs proliferation. Methods Mononuclear cells were isolated by density gradient centrifugation, and were made into 1 × 106 / ml, 1.5 × 106 / ml, 2 × 106 / ml, 2.5 × 106 / ml, 3 × 106 / ml, 3.5 × 106 / 4 × 106 / ml were inoculated into fibronectin-coated culture flasks at a density of 4 × 106 / ml. The numbers of adherent cells and colony formation of each inoculum density were compared. EPCs were identified by confocal laser scanning microscopy, flow cytometry and immunofluorescence . Results The number of adherent cells and cell colonies obtained by different inoculation densities were different. The optimal numbers of adherent cells and cell colonies obtained when inoculated at a density of 2.5 × 10 6 / ml were statistically different from other densities (P <0.05). The positive staining rate of EPCs was (1.67 ± 0.35)%, (12.63 ± 9.70)%, (66.81 ± 6.63)% respectively. The expression of CD34, AC133 and VEGFR2 in EPCs was detected by immunofluorescence. The results of adhesive ability and migration ability were (33.7 ± 1.6) and (29.2 ± 1.2) respectively. Conclusion The Ficoll density gradient centrifugation combined with adherent screening method can be used to isolate human cord blood EPCs well and obtain good cell differentiation and proliferation with 2.5 × 106 / ml density inoculation culture.