外周血单个核细胞向血管内皮祖细胞分化的体外研究

Peripheral blood mononuclear cells differentiated into endothelial progenitor cells in vitro

目的该实验从简化方法、提高得率的角度出发,试图建立直接培养单个核细胞(MNC)而得到血管内皮祖细胞(endothelialprogenitorcells,EPCs)的方法。方法抽取人外周抗凝血,用Ficoll密度梯度离心法分离得到MNC,体外对MNC进行诱导分化培养,然后选择免疫荧光、免疫组化、流式细胞记数和RT-PCR方法鉴定培养所得的细胞是否具有EPCs的特征。结果MNC在培养的第2天开始出现成簇现象;第4天细胞由圆形转变为梭形贴壁细胞,呈条索状或管状分布;第7天85%的贴壁细胞摄取Ac-LDL;第14天免疫荧光和免疫组化检测CD31、CD34、vWF、Flk-1均阳性;同时流式细胞记数检测结果显示CD31和CD34均为阳性;另外RT-PCR结果显示培养的细胞表达内皮细胞特异性基因Flt-1,Flk-1、ecNOS、tie-2等。结论用此方法培养的MNC细胞具备了内皮细胞以及干细胞双重特征,故初步认定,人外周血MNC在体外经适当条件的培养,完全可以分化成EPCs。

Objective The purpose of this study was to prove the hypothesis that EPCs be gained by directly culturing MNCs. Methods MNCs were isolated from the human peripheral anticoagulant blood sample (10-15 ml) by Ficoll-density centrifugation. The isolated cells were cultured in Medium-199 with heparin (100U / ml), 20% FBS, bovine pituitary extract (9 μg / ml). The culture flask was coated with 2% gelatin (5 μl / cm2). The experiments of cellular uptake of DiI-Ac-LDL at day 7, immunofluorescence and immunocytochemistry of CD31?CD34? Flk-1 ?vWF, flow cytometry of CD31 and CD34 at day 14, were performed to detect the cultured cells developping into EPCs in vitro. Reverse transcription-polymerase chain reaction (RT-PCR) of Flt-1, Flk-1, ecNOS and tie-2 were also observed following 14-day culture. Results 1-2×106 MNCs were isolated in one ml peripheral blood. In the first day of culture, 30% cells attached, and most of them shaped round, few spindle. Cluster attaching cells appeared in the second day. Cord-like or duct-like structure formed among much more spindle-shaped cells was observed in the day 4. DiI-Ac-LDL was took up by 85% cultured cells, and CD31?CD34?Flk-1 ?vWF were all positively stained at cytoplasm in most of attaching cells. Flow cytometric analyses showed that attaching cells were positive in CD31 and CD34, and RT-PCR revealed that attaching cells expressed Flt-1, Flk-1, ecNOS and tie-2. Conclusion The outcome of the studies suggests that there be EPCs' characteristics in the cultured cells differentiated from MNCs.