人外周血内皮祖细胞分离和诱导分化的实验研究

In vitro study on isolation and induced differentiation of human peripheral blood endothelial progenitor cells

目的探讨人外周血内皮祖细胞和单个核细胞分离培养、诱导向内皮祖细胞分化的方法与鉴定。方法采取人外周血抗凝,用淋巴细胞分离液密度梯度离心法分离外周血中的内皮祖细胞和单个核细胞,于体外以细胞因子诱导单个核细胞向内皮祖细胞分化,用免疫荧光法和流式细胞仪分离鉴定单个核细胞向内皮祖细胞分化的细胞特异性标志。结果外周血内皮祖细胞和单个核细胞共同培养3d大部分贴壁密集生长,培养6d梭形细胞增多,培养12d收获细胞进行鉴定。Dil标记乙酰化低密度脂蛋白摄取率为(70.00±11.21)%,AC133,CD31,CD34,血管内皮生长因子受体鄄2和血管性假血友病因子免疫荧光染色阳性。流式细胞仪分类计数,AC133、CD31、CD34和血管内皮生长因子受体鄄2阳性细胞分别为95.80%、87.60%、1.27%和96.40%。结论外周血循环中的内皮祖细胞和单个核细胞共同培养,诱导后者分化的内皮祖细胞具有祖细胞特性且诱导分化率高。

Objective To investigate isolated culture of human peripheral blood vascular endothelial progenitor cells （EPCs） and mononuelear cells （MNCs）, and identify that MNCs could be differentiated into EPCs. Methods Human peripheral blood EPCs and MNCs were isolated by Fieoll density gradient centrifugation. MNCs were inducted to differentiate into EPCs with eytokine in vitro. These differentiated EPCs were determined and quantified by immuno-fluoreseenee and flow eytometer. Results Most of the peripheral blood EPCs and MNCs were proliferated adhesively after being euhured for 3 days. Spindle cells were significantly increased after being euhured for 6 days. After 12 days all euhured cells were harvested for identification of EPCs markers. The uptake rate of Dil-AC-LDL was （70.00±11.21）% in cultured cells, and the cyto- plasm in most of the attaching cells were positive for AC133, CD31, CD34, vascular endothelial growth factor receptor-2 （Flk-1） and yon Willebrand factor （vWF） in immuno-fluoreseenee. Flow eytometrie analysis showed that the positive rates of AC133, CD31, CD34 and Flk-1 of attaching cells were 95.80%, 87.60%, 1.27% and 96.40% respectively. Conclusion This study suggests that EPCs differentiated from MNCs by induction have the characteristics of progenitor cells and the induction rate is high when they are eoeuhured in vitro.