目的:探讨在内皮培养条件下人外周血单个核细胞不同时段克隆成分的生物学特征。方法:11名健康志愿者,由肘静脉取血50mL,肝素抗凝,密度梯度离心法获得单个核细胞(MNCs),用含生长因子的内皮培养基接种于纤连蛋白包被的培养板中,接种后每2h去除1次未黏附细胞,共2次,然后隔日换液1次,第7d计数早期克隆。每例血样均分为2等份,1份在获得早期克隆后持续培养,直到晚期克隆出现;另1份加入无血清培养基继续培养72h,ELISA法测定上清液中血管内皮生长因子(VEGF)浓度。流式细胞技术检测细胞表面抗原CD14、CD45、CD146等的表达,直接荧光染色法测定细胞结合荆豆凝集素。结果:早期克隆中心为圆形细胞,周边是放射状排列的纺锤形细胞,再种植不能形成第2代克隆。细胞表面主要表达CD14和CD45,培养上清液中VEGF浓度明显高于晚期克隆(P<0.05)。晚期克隆呈现典型的内皮细胞特征,再种植可形成第2代克隆。细胞表面CD146表达明显增加(P<0.01),而CD45、CD14表达明显减少(P<0.001)。结论:人外周血单个核细胞在内皮培养条件下可形成早期克隆和晚期克隆,早期克隆属于单核细胞系列,可分泌VEGF但不能分化成内皮细胞,晚期克隆细胞具有内皮祖细胞的形态和生物学特征。 OBJECTIVE: To investigate the biological characteristics of clonal components in human peripheral blood mononuclear cells under different culture conditions. Methods: Eleven healthy volunteers were enrolled in this study. Mononuclear cells (MNCs) were obtained from 50 mL of blood collected from the elbow vein by heparin anticoagulation and density gradient centrifugation. The cells were seeded onto fibronectin-coated plates with growth factor-containing endothelial medium , After inoculation every 2h to remove 1 non-adherent cells, a total of 2 times, and then every other day to change fluid 1, 7d count early cloning. Each sample of blood was divided into two equal parts, one copy was obtained after the early cloning until the late clones appeared; the other one was added to the serum-free medium and cultured for 72h. The supernatant was assayed for the expression of vascular endothelial growth factor )concentration. Flow cytometry was used to detect the expression of cell surface antigens such as CD14, CD45 and CD146, and the fluorescence intensity of cells was measured by direct fluorescent staining. Results: The early cloning center was a round cell surrounded by a radial arrangement of spindle cells, which could not form the second generation clones. The cell surface mainly expressed CD14 and CD45, and the VEGF concentration in the culture supernatant was significantly higher than that in the late stage (P <0.05). Late clones show the typical characteristics of endothelial cells, replanting can form the second generation clones. Cell surface CD146 expression was significantly increased (P <0.01), while CD45, CD14 expression was significantly reduced (P <0.001). CONCLUSION: Human peripheral blood mononuclear cells can form early and late clones under the condition of endothelial culture. The early clones belong to the mononuclear cell series, which can secrete VEGF but can not differentiate into endothelial cells. The late clonal cells have the morphology and biology of endothelial progenitor cells Learn characteristics.