大鼠骨髓来源的血管内皮祖细胞的体外培养和鉴定

Culture and Identification of Rat Bone Marrow Derived-Endothelial Progenitor Cells in Vitro

目的探索大鼠骨髓来源的血管内皮祖细胞(EPCs)的体外培养、诱导分化及鉴定方法。方法采用密度梯度离心法从Wistar大鼠骨髓中分离出骨髓单个核细胞,并在诱导培养基(EGM-2MV)中培养,贴壁筛选法分离,诱导分化为EPCs;观察EPCs的生长分化过程,对其形态、表型、功能加以鉴定。结果培养3 d,细胞贴壁较为完全,第4天换液后,细胞呈现克隆样生长,第7～8天形成类似成熟血管内皮细胞形态,呈现典型的"铺路石"样外观;诱导培养第7、10天的贴壁细胞CD34、Flk-1及CDl33染色均呈阳性;荧光显微镜下观察,DIL-ac-LDL和FITC-UEA-1双荧光染色的细胞数占贴壁细胞数的75%以上。结论采用密度梯度离心法获得骨髓单个核细胞,在培养液中贴壁培养,可以扩增出具有典型特征的EPCs。

Objective To investigate methods of culturing,differentiation and identification of rat bone marrow derived-endothelial progenitor cells（EPCs）.Methods The marrow mononuclear cells were gained from Wistar rat bone marrow by density gradient centrifugation,and cultured in microvascular endothelial growth medium-2.Cells were isolated with adherence screening method and differentiated to EPCs after induction.The procedure of EPCs growing and differentiating was observed.EPCs were identified by cellular morphology and cellular phaenotype and cellular function.Results After 3 days culture,cells nearly completed adhering to culture plates.After renovating culture fluid on the 4th day,cells started to form colonies.While cultured for 7 days to 8 days,cells were differentiated into endothelial-like cells and formed typical cobblestone-like structure.On the 7th day and 10th day,adherent cells were identified positive for CD34,Flk-1 and CDl33 by immunofluorescence staining.Observation using fluorescence microscopy,the double-positive staining with DIL-ac-LDL and FITC-UEA-1 cell population was above 75 percent among adherent cells.Conclusion The bone marrow mononuclear cells are gained by density gradient centrifugation,then via adherent culture in microvascular endothelial growth medium-2,EPCs are obtained and amplified in vitro.