血管内皮祖细胞用于构建血管新生的体外三维模型

Establishment of vascular neogenetic 3D model in vitro using rabbit endothelial progenitor cells

目的采用血管内皮祖细胞(EPCs)体外构建血管新生的三维模型,证实体外获得的EPCs参与血管新生。方法(1)密度梯度离心法获取兔外周静脉血的单个核细胞,贴壁筛选法分离EPCs,于添加了Singlequotes的EBM-2培养液中扩增,对培养14 d的细胞进行免疫荧光及免疫组织化学鉴定;(2)用上述同样的方法获取EPCs,并接种于Matrigel三维基底膜胶,Singlequotes诱导,倒置相差显微镜观察血管形成。结果(1)培养7～9 d,光电显微镜可见细胞集落形成,呈现干细胞特性;贴壁细胞表达血管性假血友病因子(v WF)、血管内皮生长因子受体2(VEGFR-2)和血管内皮钙黏蛋白(VE-cadherin),呈现内皮细胞系特征;贴壁细胞用碳化青染料(DIL)标记的乙酰化低密度脂蛋白(DIL-ac-LDL)及FITC标记的I型荆豆凝集素(FITC-UEA-1)双荧光阳性,显现EPCs的生物学特性;(2)动态观察:培养2周左右,在三维胶体中形成血管结构。结论外周血来源的EPCs,经过体外诱导后,具有成血管能力,可应用于制备体外血管新生的三维模型。

Objective To study whether vascular endothelial progenitor cells could be obtained from rabbit peripheral blood and cultured for vascular neogenetic three-dimensional（3D） model in vitro. Methods The rabbit peripheral blood mononuclear cells （MNCs） were isolated by density- gradient centrifugation and amplified in endothelial basal medium supplemented with EGM-2-MV- Singlequots. Chemical fluorescence detection and immunohistochemistry were used to confirm whether the cultured cells have the biological function of endothelial cells. Vascular tube formation on Matrigel plate in vitro was observed during culturing for 14 days. Results Cultured cells exhibited colony formation and cobblestone morphology after 7 to 9 days and 4 weeks culture, respectively. Cultured cells were positively stained for yon Willebrand factor （vWF）, vascular endothelial growth factor recepter-2 （VEGFR-2） and VE-cadherin and showed endocytose UEA-1 and ac-LDL. Dynamic observation showed that in vitro vascular tube formation on Matrigel plate was seen about two weeks. Conclusion EPCs from rabbit peripheral blood mononuclear cells have an ability of vasculogenesis after indused in vitro, which can be used for establishing vascular neogenetic 3D model.