兔骨髓源血管内皮祖细胞的体外培养与鉴定

The culture and identification of endothelial progenitor cells from rabbit bone marrow

目的体外分离、培养、扩增血管内皮祖细胞（EPCs）,观察EPCs生长分化过程并对其生物学特性进行鉴定。方法采用密度梯度离心法从兔骨髓中提取单个核细胞,贴壁筛选法分离EPCs,于添加了Singlequotes的EBM-2培养液中扩增培养,对培养10d的细胞进行免疫荧光及免疫组织化学分析。结果培养4d,光电显微镜可见细胞集落形成,梭形贴壁细胞从集落中央以放射状向外周生长;培养7-10d,细胞集落相互连接,呈典型的“鹅卵石”样外观;2周左右可见细胞排列成条索状结构。贴壁细胞呈DIL-ac-LDL及FITC-UEA-1双荧光阳性,阳性率为（65.0±4.0）%;贴壁细胞表达vWF,VEGFR-2和VE-cadherin的阳性率分别为（90.0±2.1）%,（77.0±4.1）%和（78.1±8.2）%。结论骨髓中富含EPCs,成功建立了一整套骨髓源EPCs的分离、体外扩增培养的方法,且操作简便具有较好的可重复性。

Objective To study in vitro the isolation, culture and amplification of endothelial progenitor cells（EPCs） and observe the process of their growth and differentiation, then to identify their biologic character. Methods Gain mononuclearcells（MNCs）were extracted from bone marrow by density gradient centrifugation and then separated the EPCs from MNCs with the method of adherence-sieve. Analysis with immunofluorescence and immunohisrochemistry assay was carried out after ten-day culture in the EBM-2 culture medium containing Singlequotes. Results There could see the cell colony, formed by the spindle-like adherent cells radiating towards periphery on the fourth day of culture, cell colony link with each other, presenting a typical cobblestone-like appearance on the 7th to 10th day and trabs-like arrangement in two weeks or so. The spindle-like cells were reactive to DIL-ac-LDL and FITC-UEA-1, with the positive rate of （65.0±4.0）%, while the expression rates of vWF,VEGFR-2 and VE-cadherin of the spindle-like cells were （90.0±2.1）%, （77.0±4.1）% and （78.1±8.2）%, respectively. Conclusion There are rich EPCs existing in the marrows. With the method introduced above, which is simple and well repeatable, we can manage the vitro-culture amplification successfully.