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目的:从脐血中分离、培养血管内皮祖细胞,研究内皮祖细胞的生长特性和诱导分化条件。方法:应用MACS磁球抗体标记法纯化脐血中的CD133+细胞,通过流式细胞仪、免疫细胞化学、免疫荧光等技术及形态学(光镜、电镜)观察研究内皮祖细胞;将细胞接种于添加(或未添加)VEGF、bFGF、干细胞因子(SCF)的含20%胎牛血清(FBS)的IMDM培养基中,观察内皮祖细胞的生长特性。结果:分离新鲜脐血所得CD133阳性细胞占单个核细胞的(1.41±1.14)%,经流式细胞仪鉴定CD133+细胞纯度为75%-85%;将分离细胞接种于纤维连接蛋白包被的24孔板内,培养1-2h即有细胞贴壁,7-10d可见贴壁细胞呈铺路石样排列;14d后细胞出现小圆形、梭形等多样性变化,可见毛细血管管腔样结构,电镜观察可见胞浆内典型的Weibel-Palade小体;在VEGF、bFGF、SCF存在条件下,检测贴壁细胞培养14d后细胞表面抗原表达情况:与培养开始时相比,祖细胞标志CD133和CD34阳性率呈明显下降趋势,分别由(77.0±3.3)%和(93.1±4.7)%降至(1.6±2.2)%和(37.4±4.9)%,P<0.05,内皮细胞特异性标志Flk-1表达明显增加,由(22.3±3.3)%增至(94.3±4.1)%,P<0.05,同时vWF抗原呈强阳性表达,阳性率为(77.9±3.3)%。结论:根据细胞表面特异性分子标志(CD133+/CD34+/Flk-1+)可以从脐血中分离出EPCs,EPCs可在体外一定的诱导因子作用下,培养7-10d分化为成熟内皮细胞。
OBJECTIVE: To isolate and culture endothelial progenitor cells from umbilical cord blood to study the growth characteristics and differentiation conditions of endothelial progenitor cells. Methods: The CD133 + cells in cord blood were purified by macroscopical antibodies (McAbs). The endothelial progenitor cells (EPCs) were observed by flow cytometry, immunocytochemistry, immunofluorescence and morphological examination (light microscope and electron microscope). The cells were seeded in Growth characteristics of endothelial progenitor cells were observed in IMDM medium supplemented with or without VEGF, bFGF, and stem cell factor (SCF) with 20% fetal bovine serum (FBS). Results: CD133 positive cells isolated from fresh cord blood accounted for (1.41 ± 1.14)% of mononuclear cells, and the purity of CD133 + cells was 75% -85% by flow cytometry. The isolated cells were seeded on fibronectin coated 24 After culturing for 1-2 hours, cells adhered to the wall, and the adherent cells showed paving stone-like arrangement on 7-10 days. After 14 days, the cells showed small round shape and fusiform shape, showing the changes of capillary- Electron microscopy showed typical Weibel-Palade bodies in the cytoplasm. Under the condition of VEGF, bFGF and SCF, the expression of cell surface antigens was detected on the surface of adherent cells for 14 days. Compared with the beginning of culturing, the expression of CD133 and CD34 The positive rates of Flk-1 decreased significantly from (77.0 ± 3.3)% and (93.1 ± 4.7)% to (1.6 ± 2.2)% and (37.4 ± 4.9)%, respectively (22.3 ± 3.3)% to (94.3 ± 4.1)%, P <0.05. Meanwhile, the expression of vWF antigen was strongly positive with a positive rate of (77.9 ± 3.3)%. CONCLUSION: EPCs can be isolated from umbilical cord blood by cell surface specific molecular markers (CD133 + / CD34 + / Flk-1 +). EPCs can differentiate into mature endothelial cells after cultured for 7-10 days under certain inducing factors in vitro.