兔外周血内皮祖细胞的培养与鉴定

Culture and characterization of endothelial progenitor cells from peripheral blood of rabbits

目的研究兔外周血内皮祖细胞(EPCs)的分离和定向培养的方法,并对其功能进行鉴定。方法从兔股静脉插管抽取静脉血20 mL,采用淋巴细胞分离液进行密度梯度离心法分离单个核细胞,接种于人纤连蛋白包被的培养板上,分别予含血管内皮生长因子(VEGF)20 ng／mL或内皮细胞生长添加剂(ECGS)30μg／mL的M199培养基培养。培养2周后进行免疫荧光染色鉴定内皮祖细胞。结果每毫升外周血可分离1×106～2×106个单个核细胞,两组均于接种后第2～3天细胞胞体增大,有的呈多边形或梭形;第4天出现条带或栅栏状分布;第7～8天梭形细胞增多;第14天左右出现细胞集落,其特点是中央为圆形细胞,外周是梭形细胞;添加VEGF的培养基组每接种5×105个单个核细胞出现5～10个细胞集落,而添加ECGS者出现8～15个。第4周,添加VEGF的培养基组的细胞开始脱落而死亡,而添加ECGS的细胞仍持续稳定生长,呈铺路石样分布。两组在荧光显微镜下均可见双阳性的EPCs,血管性假性血友病因子(vWF)染色阳性。结论采用VEGF和ECGS均可体外培养兔外周血内皮祖细胞,且均能使其诱导分化为内皮样细胞,而ECGS较VEGF诱导效果更佳。

Objective To evaluate the efficacy of the isolation, directional culture and identification of EPCs from peripheral blood of rabbits. Methods 20 mL of blood was withdrawn from rabbits＇ femoral vein with mononuclear cells separated by density-gradient centrifugation using lymphocyte isolation and then the cells were seeded on fibronectin-coated 6-well plates and maintained in M199 with either vascular endothelial growth factor （VEGF, 20 ng/mL） group or endothelial cell growth supplements （ECGS, 30μg/mL） group. After 2 weeks, the cultured cells were identified by immunofluorescence and immunocytochemistry. Results 1×10^6-2×10^6 mononuclear cells had been isolated from 1 mL blood. At the beginning, the two groups showed the same appearances and then the cell volumes began to increase on day 2 to 3 after plating with existance of polygonal-and spindle-like cells. Approximately on day 4, the cells began to arrange in strip-line as palisade form, some aligned from head to toe. The number of spindle-like cells increased on day 7 to 8. Around 14 days after seeding, the colonies were found to be characterized by densely packed round cells surrounded by spindle-like cells. The number of colonies ranged 5-10 per 5×10^5 mononuclear cells in VEGF supplements, while in M199 with ECGS, the number was 8-15. Four weeks after plating, the cells began to shed and die in VEGF supplements. However the cells in the media with ECGS continued to proliferate and showed cobblestone appearance. Under laser scanning confocal microscopy, the differentiated EPCs were characterized as cells having dual positive for Dil-acLDL and FITC-UEA-I. EPCs were further confirmed by its expression of endothelial cell markers, vWF. Conclusions Both VEGF and ECGS can cultivate EPCs from peripheral blood of rabbits and induce EPCs to endothelial-like cells, but ECGS seems to have better indusing effect than VEGF.