人外周血单个核细胞不同克隆成分向内皮祖细胞分化的实验研究

目的:探讨从成人外周血单个核细胞中分离、培养内皮祖细胞(EPCs)的方法以及EPCs的生物学特征。方法:密度梯度离心法获得单个核细胞,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。细胞在接种后每2 h去除1次未黏附细胞共2次,然后隔日换液1次,7 d后计数早期克隆。每例血样均分为2等份,1份在获得早期克隆后进行下列实验;另1份持续培养直到晚期克隆出现进行相同实验。流式细胞技术检测细胞表面抗原表达,直接荧光染色法测定细胞结合荆豆凝集素及摄取乙酰化LDL,胶原凝胶细胞种植实验测定体外血管生成功能。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代克隆且无体外血管形成功能,细胞表面主要表达CD14和CD45。晚期克隆形态不同于早期克隆,在培养21～28 d间出现,再种植可形成第2代内皮细胞克隆,并能在胶原凝胶中形成管腔样结构。其构成细胞与早期克隆相比CD45、CD14表达显著减少(P<0.01)而CD146表达明显增加(P<0.01),2种克隆的构成细胞在结合植物凝集素和摄取乙酰化LDL方面未存在显著差异。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,晚期克隆细胞具有EPCs的形态和生物学特征。

Isolation and cultivation of different clone populations derived from human peripheral blood mononuclear cells for differentiating into endothelial progenitor cells

Objective:To investigate the methods of isolation and cultivation from adult peripheral blood mononuclear cells for the differentiation into endothelial progenitor cells(EPCs) and study the biological properties of EPCs.Method: Mononuclear cells were isolated by density-gradient centrifugation and incubated onto fibronectin-coated dishes in endothelial medium in the presence of vascular endothelial growth factor.After cultivation,cells were sequential replanted by allowing cells to adhere to tissue culture dishes during two successive 2-houre incubations in order to remove nonadherent cells.Medium was changed every other days,and the number of early clones was counted at 7 days after plating.Furthermore,every samples were divided into 2 aliquots and cells from one aliquot were cultivated continually until the late clones generated.At the same time,another aliquot was handled when the early clones can be observed after 7 days cultivation.Flow cytometry analysis was used to evaluate cells surface antigen expression and cells binding ulex europaeus agglutinin(UEA)-1 and up-taking DiI-labeled acetylated low density lipoprotein(DiI-Ac-LDL) were detected by direct fluorescence staining.The capacity of in vitro vascular genesis was assessed by plating cells onto collagen gels.Result:Early clones were identified as spindle like sprouting cells radiating from a central core of round cells.When replanted,the early clones failed to form second clone,and were devoid of the capacity of in vitro vascular genesis.Furthermore,cells derived from the early clones expressed mainly CD14 and CD45.In contrasted to early clones,the late clones emerged until 21 to 28 days after cultivation and exhibited typically endothelial cells properties.Remarkably,cells originated from late clones had the ability to form second endothelial clones after replanted and generated tube-like structures when seeded onto collagen gels.In addition to the clearly morphological differences,cells derived from late clones expressed obviously increased CD146(P<0.01) and reduced CD45 and CD14(P<0.001).There were no differences about cell binding UEA-1 and up-taking DiI-Ac-LDL between early and late clones.Conclusion:Under endothelial cultivating conditions,human peripheral blood mononuclear cells can generate early and late clones.Cells derived from early clones are relevant to monocyte linage and have not the ability to differentiate into endothelial cells.Alternatively,cells originated from late clones exhibit morphologically and biologically characteristics of EPCs.