Zheng Weihong, Wan Yafeng, Ma Xiaopeng, Li Xingrui, Yang Zhifang, Yin Qian, Yi Jilin
Department of General Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
J Huazhong Univ Sci Technolog Med Sci. 2010 Feb;30(1):18-24. doi: 10.1007/s11596-010-0104-6. Epub 2010 Feb 14.
Two isolation methods for sorting of endothelial progenitor cells (EPCs): from peripheral blood mononuclear cells (PBMCs) and CD133(+) enriched cells were compared, by defining the cell morphology, phenotype, reproductive activities and function in vitro, to provide a reference for clinical application of EPCs. PBMCs from healthy subjects were used either directly for cell culture or for CD133(+) sorting. The two groups of cells were cultured in complete medium 199 (M199) for 7 to 14 days and the phenotypes of EPCs were analyzed by FACS. The proliferation of differentiated EPCs was studied by MTT assay, and the VEGF concentration was measured using an ELISA kit. ECM gel experiment and migration assay were performed in vivo. The results showed that PBMCs produced more colony-forming units (CFU) than CD133(+) enriched cells from the same volume of blood (P<0.01). From day 7 to 14, the two groups showed decreased expression of hematopoietic stem cell markers and increased level of endothelial markers, but CD144(+) cells in CD133(+) group were less than in PBMCs group (P<0.01). PBMCs group secreted more VEGF than CD133(+) group on the day 7 (P<0.01). As compared with CD133(+) group, PBMCs group had more potent potential of proliferation and vascularization in vitro. It was concluded that CD133(+) sorted cells showed a lower capacity of differentiation, secretion, proliferation and vascularization in vitro, suggesting that CD133-negative cells may be a preferential way to get EPCs for clinical therapy.
比较了两种用于分选内皮祖细胞（EPCs）的分离方法：从外周血单个核细胞（PBMCs）中分离和从富集的CD133(+)细胞中分离，通过定义细胞形态、表型、体外增殖活性和功能，为EPCs的临床应用提供参考。来自健康受试者的PBMCs要么直接用于细胞培养，要么用于CD133(+)分选。将两组细胞在完全培养基199（M199）中培养7至14天，并用流式细胞术分析EPCs的表型。通过MTT法研究分化的EPCs的增殖情况，使用ELISA试剂盒测量VEGF浓度。在体内进行细胞外基质凝胶实验和迁移试验。结果显示，相同体积血液中，PBMCs产生的集落形成单位（CFU）比富集的CD133(+)细胞更多（P<0.01）。从第7天到第14天，两组造血干细胞标志物表达降低，内皮标志物水平升高，但CD133(+)组中的CD144(+)细胞少于PBMCs组（P<0.0１）。在第7天，PBMCs组分泌的VEGF比CD133(+)组更多（P<0.01）。与CD133(+)组相比，PBMCs组在体外具有更强的增殖和血管生成潜力。结论是，分选的CD133(+)细胞在体外显示出较低的分化、分泌、增殖和血管生成能力，这表明CD133阴性细胞可能是临床治疗中获取EPCs的优先途径。
