用CD133免疫磁珠分离脐血内皮祖细胞的实验研究

Isolation of endothelial progenitor cells from cord blood with CD133 immunomagnetic sorting

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。

AIM： To isolate, purify and differentiate endothelial progenitor cells from cord blood in vitro and to study their biological characteristics. METHODS： CD133^＋ cells were selected from fresh cord blood mononuclear cells （MNC） by magnetic activated cell - sorting system （ MACS）. EPC was studied by flow cytometry, immunocytochemistry and immunofluorescence staining, lsolated cells were cultured in IMDM medium supplemented with or without VEGF, bFGF, SCF. RESULTS： The percentage of CD133^＋ cells of cord blood MNC was （1.41 ± 1.14）%, and purity was 75% - 85% （FACS method）. CD133^＋ cells were grown on fibronectin - coated chandrer slides in the presence of VEGF, bFGF, SCF. Within 1 - 2 hours of culture cells became adherent. On clay 7- 10, the adherent cells displayed a typical “cobblestone” morphology. After 14 days of culture, the adherent cells revealed a heterogeneous cell population, comprising small- sized round cells, spindle- like cells and formed tubelike structure. Weibel- Palade bodlies were shown on the transmission electron microscopy photomicrographs. Compared with the original, cell markers CD133 and CD34 decreased significantly （77.0% ± 3.3% to 1.6% ± 2.2% and 93.1% ± 4.7% to 37.4% ± 4.9%, P 〈 0.05）, while Flk - 1 increased significantly （from 22.3% ± 3.3% to 94.3% ± 4.1%, P 〈 0.05） after 14 days of culture with VEGF, bFGF, SCF. Tbe vWF was strongly expressed （77.9% ± 3.3% ） on the 14th day later. CONCLUSION： Vascular endothelial progenitor cells were isolated from cord blood with specific expression of CD133/CD34/Flk - 1 . With the stimulation of the growth factors, seven - ten days after culture EPCs could be turned to endothelial cells.