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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。
OBJECTIVE: To elucidate the method of isolating and culturing EPCs from human umbilical cord blood mononuclear cells and to observe the expression of endothelial cell specific antigen during the proliferation and differentiation of EPCs in vitro, so as to lay a foundation for further study on the clinical application of EPCs in ischemic retinal diseases. Methods: Umbilical cord blood mononuclear cells were obtained by density gradient centrifugation, induced, differentiated and expanded in vitro. EPCs derived from umbilical cord blood were identified by immunohistochemistry and flow cytometry. In our study, The expression of CD34, VEGFR-2, EPCs-specific antigen CD133, and endothelial cell-specific antigen CD31 and factor VIII-related antigen (vWF) were analyzed. At the same time, we further identified cells by analyzing the cellular uptake of acLDL and binding to lectins. Results: Umbilical cord blood mononuclear cells were mainly spindle cells in the early culture stage, and gradually appeared the appearance of paving stones. Flow cytometry analysis of adherent cells showed that the positive rate of CD133, CD34 and VEGFR-2 Respectively. The expression rates of CD31 and vWF were 92.7% ± 2.2% and 73.3% ± 4.2% respectively by immunohistochemical staining. The results of immunofluorescence staining Indicating that 83.0% ± 4.3% of adherent cells were positive for DiI-acLDL and FITC-UEA-I staining. CONCLUSION: This experiment demonstrates that EPCs can be isolated and cultured from cord blood mononuclear cells in vitro under specific culture conditions, which lays the foundation for further research on the relationship between EPCs and retinal neovascularization and EPCs for the treatment of ischemic fundus.