人脐血内皮祖细胞体外培养和鉴定的研究

目的:阐述从人脐血单核细胞分离培养EPCs的方法,并观察EPCs体外增殖分化过程中内皮细胞特异性抗原的表达,为进一步研究EPCs在缺血性视网膜疾病的临床应用奠定基础。方法:采用密度梯度离心方法获得脐带血单个核细胞,体外进行诱导、分化和扩增,通过免疫组化和流式细胞仪分析等技术对脐血来源的EPCs进行鉴定,在我们的研究中主要分析CD34,血管内皮细胞生长因子受体-2(VEGFR-2),EPCs特异性抗原CD133,以及内皮细胞特异性抗原CD31和第八因子相关抗原(vWF)的表达情况。同时我们通过分析细胞摄取乙酰化低密度脂蛋白(acLDL)和结合植物凝集素的能力进一步鉴定细胞。结果:脐带血单个核细胞在培养早期主要为梭形细胞,逐渐呈现铺路石样外观;在细胞培养至第7d,贴壁细胞流式细胞仪分析显示,CD133, CD34和VEGFR-2的阳性率分别为17.8%±3.7%, 22.1%±4.4%和81.5%±5.0% ;免疫组化染色结果显示CD31、vWF的表达率分别为92.7%±2.2%和73.3%±4.2%;免疫荧光染色结果表明83.0%±4.3%的贴壁细胞DiI-acLDL和FITC-UEA-I染色均阳性。结论:本实验证明在体外特定的培养条件可以从脐带血单个核细胞中分离培养出EPCs,为进一步研究EPCs与视网膜新生血管的关系以及EPCs治疗缺血性眼底的临床研究奠定了基础。

Culture and identification of endothelial progenitor cells from human umbilical cord blood

AIM:To elucidate a simple method for isolating endothelial progenitor cells(EPCs)from human umbilical cord blood mononuclear cells and observe the endothelial cell-specific expression profile during proliferation and differentiation in vitro.METHODS:Human umbilical cord blood were isolated by Percoll density gradient centrifugation from human cord blood and cultured in vitro.The adherent cells were then identified by immunohistochemical staining and flow cytometric analysis.CD34,vascular endothelial growth factor receptor-2(VEGFR-2),EPCs specific antigen CD133,as well as endothelial cell specific markers CD31 and vWF,were used in our study.The cells were further characterized by acetylated LDL(acLDL)uptaking and lectin binding by direct fluorescentstaining.RESULTS:During culture,the attached cells exhibited spindle-shape in early stage,and gradualy display endothelium-like cobblestone morphology with outgrowth.On day 7,flow cytometric analysis showed that the positive staining rate of attached cells for CD133,CD34 and VEGFR-2 were 17.8%±3.7%,22.1%±4.4%and 81.5%±5.0%respectively.While,immunohistochemical staining showed that the adherent cells were positive to CD31 and vWF at the rate of 92.7%±2.2%and 73.3%±4.2%respectively.By direct fluorescentstaining,we observed that 83.0%±4.3%of the attached cells were double positive for Dil-acLDL and FITC-UEA-I.CONCLUSION:EPCs can be separated from human cord blood under certain conditions in vitro.This observation may provide a basis for study of relationship between EPCs and retinal neovascularization,as well as further clinical application of EPCs in ischemic retinal lesions.