兔外周血源的血管内皮祖细胞体外扩增研究

目的探讨从兔外周血单个核细胞中富集血管内皮祖细胞（endothelial progenitor cells,EPCs）并在体外诱导培养和扩增的方法,试图证实外周血是获得血管内皮祖细胞理想来源之一。方法密度梯度法分离兔外周血单个核细胞（MNCs）,采用专用的EGM-2-MV完全培养基对单个核细胞进行诱导分化培养,动态观察细胞生长过程,并应用免疫组织化学和细胞荧光化学法分别鉴定培养细胞的内皮细胞表面标记和生物学功能。结果外周血单个核细胞经专用培养基体外诱导后,4 d左右可见多数细胞贴壁生长,9 d后可见位于中间的细胞聚集成团,4周后呈铺路石样内皮细胞形态。培养12 d细胞能表达的内皮细胞表面抗原,包括Ⅷ因子（VWF）,血管内皮生长因子受体（VEGFR-2）和钙粘素（VE-cadherin）等;并能特异性吸附异硫氰酸荧光素（FITC）标记的荆豆凝集素（UEA-1）,能内吞乙酰化低密度脂蛋白（ac-LDL）,具备内皮细胞的功能。结论兔外周血中存在具有增殖分化潜能的EPCs,经过特定的体外诱导培养可以收集和扩增。

Culture and Proliferation of Rabbit Endothelial Progenitor Cells from Peripheral Blood

Objective To study whether endothelial progenitor cells could be obtained from rabbit peripheral blood and could be proliferated and cultured in vitro.Methods Total rabbit peripheral blood mononuclear cells(MNCs) were isolated by density-gradient centrifugation and were suspended in endothelial basal medium supplemented with EGM-2-MV-SingleQuots.Process of cell growth was observed.Immunohistochemistry was utilized to analyze the endothelial cell markers such as VWF,VEGFR-2 and VE-cadherin expressed on the surface of the cultured cells,Chemical fluorescence detection was performed to confirm whether the cultured cells have the biological function of endothelial cells by detecting dual binding of UEA-1 and ac-LDL,which consistents with endothelial lineage cells.Results Cultured cells exhibited colony formation and cobblestone morphology after 12 days or 4 weeks culture respectively.In cultured cells were positively stained for VWF,VEGFR-2 and VE-cadherin can be expressed by staining and endocytose UEA-1 and ac-LDL conld be shown too.Conclusion EPCs present in circulation and can be obtained from rabbit peripheral blood mononuclear cells.It has be shown that EPCs can be proliferated and harvested under certain condition in vitro.