脐血、外周血内皮祖细胞分化成内皮细胞的实验研究

Inducing cord blood and peripheral blood derived EPCs into endothelial cells in vitro

目的探讨人的脐血、外周血内皮祖细胞(endothelialprogenitorcells,EPCs)体外分离、纯化、诱导扩增和分化为内皮细胞的可行性,并检测其表型和功能。方法新鲜脐血和健康成年人的外周血,使用Ficoll密度梯度离心法得单个核细胞,在M199培养基中体外培养,3d后去除悬浮细胞,继续培养,诱导EPCs增殖和分化。流式细胞仪检测EPCs标志CD34和内皮细胞特异性标志CD31表型,RTPCR检测ecNOS,flk1/KDR基因水平表达,免疫组化验证蛋白水平表达,并进一步通过NO活性的变化检测内皮细胞的功能。结果流式细胞仪检测,外周血单个核细胞(peripheralbloodmononuclearcells,PBMC)刚分离时,CD34阳性表达率为(1.1±0.8)%,培养3d后为(16.9±6.2)%。细胞形态观察发现,刚分离的单个核细胞呈圆形,形态小,3d后有明显集落形成,7d后梭形细胞线样排列,随培养时间增加,细胞形态逐渐变大,呈现出典型铺路石样改变。脐血单个核细胞(umbilicalcordbloodmononuclearcells,CBMC)和PBMC培养10d后,CD31阳性表达率分别为(76±17)%和(82±9)%。RTPCR检测有内皮细胞特异性成分ecNOS,flk1/KDR的表达。免疫组化染色,细胞膜和细胞浆中有弥漫性棕色出现,呈阳性反应,证实了蛋白水平的表达。培养10d的贴壁细胞随着VEGF浓度增加,NO生成增加,具有内皮细胞的功能。结论脐血,外周血EPCs体外分离,纯化,诱导培养后的贴壁细胞表型检测,大部分细胞具有内皮系标志物,并具有产生NO功能。

AIM To investigate the feasibility of inducing human umbilical cord blood and peripheral blood derived EPCs into endothelial cells in vitro, and to test their phenotype and function. METHODS Mononuclear cells were isolated from human umbilical cord blood and peripheral blood by density gradient centrifugation, and cultured in M-199 medium. After 3 days, non-adherent ceils were removed, the culture was maintained. The EPCs specific surface mark CD34 and endothelial specific surface marker CD3! were assessed by fluorescence activated cell sorter （FACS） analysis. Endothelial specific ecNOS and flk-1/KDR were detected by immunocytochemistry staining, reverse transcription-polymerase chain reaction （ RT-PCR）, respectively. The endothelial function of the ceils was determined by measuring nitric oxide （NO） production in response to different concentrations of VEGF. RESULTS： CD34 positive ceils were only （ 1.1 ± 0.8 ） % when freshly isolated PBMC, and increased to （ 16.9 ± 6.2 ） % after 3 days. When CBMC, PBMC were cultured in medium, a number of cell clusters appeared at 3 d, and spindle-shaped ceils were observed and linear cord-like structures were formed at 7 d. Gradually, the attached （AT） ceils exhibited cobblestone morphology, which was characteristic of endothelial ceils. At 10 d, CD31 positive ceils were （76 ± 17）% and （82 ± 9）% in cultured CBMC and PBMC, respectively. The expression of EC marks of ecNOS and flk-1/KDR was detected by RT-PCR and further confirmed by immunocytochemistry staining. NO production increased with incremental dose of VEGF. CONCLUSION When isolated and cultured in special medium, PBMC and CBMC show EC-like morphology, express the marks of EC lineage and have EC function.