人循环内皮祖细胞不同克隆成分的体内外血管生成功能不同

目的探讨成人外周血循环来源的内皮祖细胞不同克隆成分表型特点及体内外血管生成差异。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维连接蛋白包被的培养板中。7d后计数早期克隆并进行下面实验,另1份持续培养直到晚期克隆出现进行相同实验。流式细胞术检测细胞表面抗原,间接荧光染色法鉴定细胞表达假血友病因子。胶原凝胶细胞体外种植及裸鼠体内移植实验分别测定体外及体内血管生成功能。结果早期克隆再种植不能形成第二代克隆且无体内外血管形成功能,细胞表面主要表达CD14和CD45。晚期克隆在培养21~28d间出现,再种植可形成第二代内皮细胞克隆,并能在体外和裸鼠体内胶原凝胶中形成管腔样结构,细胞表达CD45和CD14显著减少(P<0.001)而CD146明显增加(P<0.01)。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,只有晚期克隆表现出干/祖细胞和内皮细胞双重表型特征并具有体内外血管生成功能。

In Vitro and in Vivo Angiogenic Capacity Analysis on Different Clonal Endothelial Progenitor Cell Populations Derived from Human Circulating Blood

Aim To explore the in vitro and in vivo angiogenic capacity and phenotypic properties about different clonal endothelial progenitor cell populations derived from human peripheral circulating blood. Methods 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.The number of early clones was counted at 7 days and cells from another aliquot were cultivated continually until the late clones generated.Flow cytometry analysis was used to evaluate cells surface antigen expression and von willebrand factor(vWF),the endothelial cells marker was detected by indirect fluorescence staining.The capacities of in vitro and in vivo vascular genesis were assessed by plating cells onto collagen gels and implanting the cellularized gel into nude mice. Results Early clones failed to form second clone and were devoid of the capacity of in vitro and in vivo vascular genesis.Furthermore,cells derived from the early clones expressed mainly CD14 and CD45.In contrast 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 or transplanted into nude mice.In addition to the clearly morphological and functional differences,cells derived from late clones expressed obviously increased CD146(P<0.01) and reduced CD45 and CD14(P<0.001). Conclusions Under endothelial cultivating conditions,human peripheral blood mononuclear cells can generate early and late clones.Only cells originated from late clones exhibit double phenotypes of stem/progenitor and endothelial cells with the capacity of in vitro and in vivo vasculargenesis.