摘要
培养和鉴定从鼠骨髓中分离的内皮祖细胞（EPCs），观察其在体外增殖、分化过程中相关细胞表型的变化。采用密度梯度离心方法获得鼠骨髓单个核细胞，用EGM-2培养基诱导其分化，于培养4d、7d、10d、14d、21d后用免疫荧光双标技术（CD34、VWF和FLK1）鉴定EPCs，用MTT法分析EPCs的增殖情况。于诱导分化后的1d、4d、7d、10d、14d、21d，用蛋白免疫印迹法检测VWF蛋白的水平，用实时聚合酶链式反应法检测VEGFR 2（FLK1）、VWF的mRNA表达变化，并与未诱导培养的细胞进行比较。鼠骨髓单个核细胞经诱导培养，4d细胞开始贴壁，7d细胞大多变为梭形，排列成铺路石样。MTT结果表明，10d时EPCs达到增殖高峰（P〈0．05）；免疫染色表明，培养7d的细胞约90％呈VWF—CD34双荧光阳性；蛋白免疫印迹法检测结果表明，培养1d的细胞无VWF表达，培养4d后表达逐渐增强，10～14d达到高峰，21d后减弱；实时聚合酶链式反应法检测结果表明，培养1d的细胞FLK1和VWF的mRNA表达低（P〈0．01），4d后表达逐渐增强，14d达到高峰，21d后减少，未诱导细胞无表达。试验成功地从鼠骨髓单个核细胞中分离培养出EPCs，在其体外扩增分化为成熟内皮细胞的过程中，P0～P3在1～21d期间FLK1和VWF等内皮细胞标志蛋白表达逐渐增强，P3在21d后略有下降。该细胞可作为构建血管组织工程支架的较为理想的种子细胞。
To elucidate a simple method of isolating endothelial progenitor cells （EPCs） from rat marrow monocytes （MNCS） and observe the endothelial cell specific expression profile during proliferation and differentiation in vitro. MNCS were isolated from rat marrow using ficoll density gradient centrifugation, induced, proliferated and differentiated in EGM-2 culture medium. The EPCs were identified by immunofluorescent staining （CD34,VWF diplmark and FLK1） at 4d,7d, 10d, 14d,21d. The proliferation of EPCs was evaluated by MTT, and endothelial cell specific markers （VWF,FLK1） were determined with western blotting and real-time PCR at ld,4d,7d, 10d, 14d, 21d. During culturing process, the ceils became spindle shaped and displayed endothelium-like cobble-stone morphology, with outgrowth at 4d and 7d. Result of MTT indicated that the number of EPCs increased to peak at 10d （P 〈 0.05）. Percentage of the cells that were positive for CD34/VWF was 90% at 7d. Expression for VWF at ld was not found with western blotting, and however gradually increased at 4d and reached to the top during 10d to 14d. The resuhs with real-time PCR indicated that the expression of VWF and FLK1 mRNA within non-induced cells was not found, gradually increased at 4d and reached to top at 14d, the low-expression at ld was significantly different from the other ones （P 〈 0.01）. （These data demonstrated that EPCs could be isolated from rat marrow monocytes （MNCS） and cultured in vitro. The EPCs could be used as seeding ceils in vascular tissue engineering.
出处
《中国生物医学工程学报》
CAS
CSCD
北大核心
2008年第3期443-450,共8页
Chinese Journal of Biomedical Engineering
关键词
内皮祖细胞
内皮细胞
血管新生
组织工程
endothelial progenitor ceils
endothelial ceils
angiogenesis
tissue engineering
