赵梦蝶, 查从亮, 江浩, 项平. 人脐带血内皮祖细胞的分离、培养与鉴定[J]. 蚌埠医学院学报, 2016, 41(9): 1121-1124. DOI: 10.13898/j.cnki.issn.1000-2200.2016.09.001
    引用本文: 赵梦蝶, 查从亮, 江浩, 项平. 人脐带血内皮祖细胞的分离、培养与鉴定[J]. 蚌埠医学院学报, 2016, 41(9): 1121-1124. DOI: 10.13898/j.cnki.issn.1000-2200.2016.09.001
    ZHAO Meng-die, ZHA Cong-liang, JIANG Hao, XIANG Ping. Study on the separation,culturation and identification of human umbilical cord blood progenitor cells[J]. Journal of Bengbu Medical College, 2016, 41(9): 1121-1124. DOI: 10.13898/j.cnki.issn.1000-2200.2016.09.001
    Citation: ZHAO Meng-die, ZHA Cong-liang, JIANG Hao, XIANG Ping. Study on the separation,culturation and identification of human umbilical cord blood progenitor cells[J]. Journal of Bengbu Medical College, 2016, 41(9): 1121-1124. DOI: 10.13898/j.cnki.issn.1000-2200.2016.09.001

    人脐带血内皮祖细胞的分离、培养与鉴定

    Study on the separation,culturation and identification of human umbilical cord blood progenitor cells

    • 摘要: 目的:分离、培养、鉴定人脐带血内皮祖细胞(EPCs),为获取大量血管内皮祖细胞提供方法。方法:脐带血采集后,采用6%羟乙基淀粉沉降法和密度梯度离心法获取脐带血单核细胞,再将单核细胞接种于铺设有纤维连接蛋白的培养瓶中,经过体外诱导、培养、分化,完成传代扩增;通过免疫组织化学、免疫荧光染色、流式细胞术对细胞进行鉴定。结果:细胞培养第5天呈现集落样生长,单个细胞呈圆形或梭形,2周后细胞长满瓶底,呈鹅卵石样外观。免疫组织化学检测显示,CD31兔抗人单克隆抗体阳性率91.5%,Anti-Von Willebrand factor antibody(vWF)相关抗原兔抗人单克隆抗体的阳性表达率为72.4%;细胞免疫荧光染色检测显示,吞噬Dil标记的乙酰低密度脂蛋白(+),发出红色荧光;结合FITC标记的荆豆凝集素Ⅰ(+),发出绿色荧光;双染(+),呈黄色荧光。流式细胞术检测CD34阳性率93%,血管内皮细胞生长因子受体2阳性率88.5%,CD133阳性率84.8%。结论:从脐带血中可获取大量CD34+、VEGFR-2+及CD133+血管内皮祖细胞。

       

      Abstract: Objective: To study on the separation,culturation and identification of human umbilical cord blood progenitor cells from human cord blood progenitor cells (EPCs),and provide a method to obtain a large number of endothelial progenitor cells.Methods: After cord blood were collected,with methods of 6% hydroxyethyl starch sedimentation and density gradient centrifugation,we obtained cord blood mononuclear cells.And then the cells were seeded in culture flasks laid fibronectin.After the cells were induction culturation,and differentiation in vitro,passage and amplification of the cells were completed;the cells were identified by immunohistochemistry,immunofluorescence,flow cytometry.Results: The cell culture showed colony-like growth at the first five days,and each single cell showed round or spindle-shaped.Two weeks later,the cellscovered the bottom of culture dish,and showed cobblestone-like appearance.Immunohistochemistry got 91.5% positive rate of CD31,72.4% positive rate of vWF;immunofluorescence staining told us that the cell emited red fluorescence,and phagocytose DiI-acLDL(+);but when the cells dyed with FITC marked by UEA-Ⅰ(+) they emited green fluorescence,and when the cells were double stained,they emited the yellow fluorescence.Flow cytometry analysis showed that the rate of CD34-positive cells was 93%,VEGFR-2 positive cells was 88.5%,and CD133 positive cells was 84.8%.Conclusions: CD34+、VEGFR-2+ and CD133+ endothelial progenitor cells can be largly obtained from the umbilical cord blood.

       

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