人脐血来源内皮祖细胞的纯化鉴定及定向分化的研究

Purification, identification and oriented differentiation of endothelial progenitor cells from human umbilical cord blood

目的检测CD133+血管内皮祖细胞(EPC)的细胞表面标志物,鉴定其生物学特性。方法采用免疫磁珠法分离人脐血EPC,用EGM-2MV培养基[含表皮生长因子(EGF)、血管内皮生长因子(VEGF)、成纤维细胞生长因子(FCF)2等具有诱导分化作用的细胞因子]进行体外培养。应用流式细胞术、免疫组织化学等方法检测CD133+细胞的比例、原代EPC的生长曲线及生长特性。透射电镜查找Weibel-Palade小体。同时没计EPC裸鼠成瘤实验,观察瘤体生长及血管增生情况,以鉴定EPC的生物学特性。结果CD133+细胞在免疫磁珠分离前后的比例各为0．91％及85．52％。EPC原代培养时细胞形态正常、密度均匀,前3 d为相对抑制期,此后快速增殖,10 d后达80％-90％融合。EPC生长曲线显示,接种后5 d内细胞数量无明显变化,从第7天开始明显增加,第17天达高峰。光学显微镜下可见,原代EPC贴壁后呈梭形,接种后7 d数量明显增加,呈克隆状生长。透射电镜观察到,细胞膜伸出许多伪足丝,基底膜呈多层;细胞质内可见一种短棒状小体,含平行管状的内部结构,即Weibel-Palade小体,确定其为EPC。裸鼠成瘤实验可见EPC组肿瘤瘤体及血管数量大于或多于对照组;抗人血管性假血友病因子(vWF)免疫荧光染色显示,大量EPC参与肿瘤血管生成。结论本实验分离培养的CD133+细胞具有分化为成熟血管内皮细胞的能力,即为EPC。裸鼠成瘤实验初步证明EPC参与血管重建,具有促进血管新生、加速缺血组织血管化的作用。

Objective To explore the endothelial progenitor cell markers and biological characteristics of human CD133^＋ umbilical cord blood ceils（EPC）. Methods CD133^＋ ceils were enriched from human umbilical cord blood by immunomagnetic sorting, and cultured with EGM-2MV medium containing epidermal growth factor, vascular endothelial growth factor and fibroblast growth factor 2. The percentage of CD133^＋ ceils in cord blood monocytes, the growth curve and growth characteristics of primary EPCs were measured by flow cytometry and immunochemistry method. Weibel-Palade body was observed with transmission electron microscope. The mixture of EPCs and human stomach cancer cell line GC7901 were injected into athymic mice to observe the tumor growth and vascularization. Results The percentage of CD133^＋ ceils in cord blood monocytes was 0.91% , and after sorting, the percentage of CD133^＋ ceils was raised to 85.52%. The cultured ceils showed a typical spindle-shaped morphology in 3 post-culture days（PCD） and areas of clusters of cobblestone-like cells in 10 PCD. The number of EPC increased from 7 PCD on, peaked on 17 PCD. Obvious amplification and clone-like growth on 7 PCD were observed by light microscope. Typical Weibel-Palade body was observed in the ceils under transmission electron microscope . Tumor forming experiment in athymic mice showed that the tumor size of EPC group was larger than that of control with smaller necrosis area and more and larger blood vessels. Immuno-fluorescent staining showed many human vWF antigen-positive endothelial ceils being involved in the tumor vascularization. Conclusion Immunomagnetic sorting can efficiently enrich EPC from human umbilical cord blood. Our data support that the EPC may contribute to angiogenesis, speed up vascularization of ischemic tissue.