CD31免疫磁珠分选人脂肪内皮祖细胞的培养及鉴定

Culture and identification of endothelial progenitor cells from human adipose tissue with CD31 immunomagnetic beads

背景:内皮祖细胞是成熟内皮细胞的前体细胞,可迁移到缺血组织,分化为成熟内皮细胞,发挥血管新生作用。在各种缺血性疾病具有广阔的应用前景,但其生物学特性及鉴定方法仍存在争议。目的:探讨从人脂肪组织中分离培养内皮祖细胞的方法,并进行鉴定,为移植治疗缺血性疾病提供足量的细胞来源。方法:采用酶消化法从人脂肪组织中分离培养出脂肪基质血管细胞(stromal vascular cells,SVF),再通过免疫磁珠分选出CD31^+细胞,体外扩增培养得到内皮祖细胞,并通过细胞形态、细胞生长增殖能力,细胞表型及细胞的功能多方面来完成鉴定。结果与结论:①免疫磁珠分选后的CD31+细胞,体外扩增培养呈典型的铺路石样形态,且其保持一定的生长增殖潜能;②第5代CD31^+细胞流式分析结果显示:CD31、CD34、VEGRR2、CD146和CD133表达量分别为98.84%、97.21%、64.07%、98.42%和2.55%,但几乎不表达造血干细胞表面标志CD45(1.1%);③免疫荧光染色结果表明:CD31^+细胞可摄取乙酰化低密度脂蛋白(Dil-ac-LDL)并可结合荆豆凝集素(FITC-UEA-Ⅰ)。并且,CD31+细胞在Matrigel基质胶中可形成管腔样结构,具有体外成血管能力;④结果提示,通过酶消化法结合免疫磁珠分选法从人脂肪组织中分离培养内皮祖细胞是可行的。

BACKGROUND:Endothelial progenitor cells are precursor cells of mature endothelial cells,which can migrate toischemic tissues and differentiate into mature endothelial cells,and then play an important role in vascular remodeling.Endothelial progenitor cells have wide application prospects in various ischemic diseases,but the biologicalcharacteristics and identification methods are still controversial.OBJECTIVE:To investigate the methods of isolation and culture of endothelial progenitor cells from the humanadipose tissue and to identify their biological features,in order to provide a sufficient source of cells for ischemicdiseases.METHODS:Stromal vascular fraction cells were isolated from the human adipose tissue by enzymatic digestion,CD31+cells were selected using immunomagnetic beads,and then cultured in endothelial basal medium-2supplemented withthe EGM-2-MV-SingleQuots.Endothelial progenitor cells were identified through detection of morphology,cell markersand cell functions.RESULTS AND CONCLUSION:(1)CD31+cells were selected by immunomagnetic beads and then cultured andamplified in vitro,which displayed typical cobblestone-like morphology,and they maintain their proliferative ability.(2)Flow cytometry results showed that the CD31+cells expressed CD31(98.84%),CD34(97.21%),VEGRR2(64.07%),CD146(98.42%)and CD133(2.55%),but hardly expressed CD45(1.1%),a hematopoietic stem cell marker.(3)TheCD31+cells were also found to incept Dil-ac-LDL and exhibit lectin binding capability.Furthermore,a lumen-like structurewas formed in Matrigel,which has the ability of angiogenesis in vitro.To conclude,these results suggest that it is feasibleto isolate and culture endothelial progenitor cells from the human adipose tissue by enzymatic digestion combined withimmunomagnetic bead sorting.