外周血内皮祖细胞体外培养分化研究

Culture and Differentiation of Endothelial Progenitor Cells from Peripheral Blood in Vitro

目的从人外周血中分离、培养和鉴定内皮祖细胞(EPCs),并观察其在体外增殖分化过程中各种细胞表型的变化。方法采用密度梯度离心方法获得外周血单个核细胞,体外进行诱导、分化和扩增,于培养第7天选择免疫荧光(DiI-acLDL/FITC-UEA-Ⅰ)鉴定EPCs,并且用流式细胞仪和RT-PCR方法观察上述细胞在第0、4、10和21天的CD34、CD31、KDR和eNOS的表达变化。结果外周血单个核细胞在培养过程中出现梭形贴壁和铺路石样等形态;第7天免疫荧光染色表明,约70%的细胞呈双荧光阳性;流式细胞仪分析显示,CD31和KDR表达在体外培养过程中逐渐升高,至第21天分别达到(72.1±11.2)%和(81.0±12.5)%,而CD34在第10天达到高峰(38.0±13.4)%后,第21天下降为(28.3%±12.2)%;RT-PCR结果表明,第4、10和21天eNOS的表达逐渐增强。结论本试验成功从外周血单个核细胞中分离培养出EPCs,在其体外扩增分化为成熟内皮细胞的过程中,CD31、KDR和eNOS等内皮细胞标志表达逐渐增强,而CD34的表达在内皮祖细胞的成熟分化过程中略有下降。

Objective To elucidate a simple method of isolating endothelial progenitor cells （EPCs） from human peripheral blood monocytes （PBMCs） and observe the endothelial cell-specific expression profile during proliferation and differentiation in vitro. Methods PBMCs were isolated by ficoll density gradient centrifugation from peripheral blood, cultured and then identified by immunofluorescent staining （DiI-acLDL/ FITC-UEA- I ） at d7. Endothelial cell-specific markers （CD34,CD31 ,KDR,eNOS） were determined with flow cytometry analysis and RT-PCR at dO, d4,d10,and d21. Results During culturing, cells became spindle-shaped and displayed endothelium-like cobblestone morphology with outgrowth. The double positive for DiI-acLDL/ FITC-UEA- I accounted for 70% of the attached cells at d7. Expressions of CD31, KDR and eNOS were significantly increased during the diffe-rentiation course, while CD34 was decreased at d21 as compared with d10 （38.0% ± 13.4% vs 28.3% ±12.2% ）. Conclusion These data demonstrated that EPCs could be isolated and cultured from PBMC fraction, and during culturing endothelial cell-specific marker expressions, such as CD31, KDR and eNOS were potently enhanced, while CD34 was somewhat decreased in differentiating into mature endothelial cells.