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目的优化选择猪骨髓来源内皮祖细胞(endothelial progenitor cell,EPC)体外培养条件,为后续研究奠定基础。方法从猪髂骨抽取骨髓,利用密度梯度离心法分离得到单个核细胞(MNC),体外培养分化为EPC。在其他培养条件相同的前提下,分别比较不同的细胞接种密度(2×103/cm2、5×103/cm2、1×104/cm2、2×104/cm2),不同基础培养液(EGM、M199、DMEM),不同FBS浓度(5%、10%、20%、30%),以及血管内皮细胞生长因子(VEGF)与不同细胞因子[碱性成纤维细胞生长因子(bFGF)、基质细胞衍生因子(SDF)、胰岛素样生长因子(IGF)和表皮生长因子(EGF)]组合(VEGF+bFGF、VEGF+SDF、VEGF+bFGF+SDF、VEGF+bFGF+IGF+EGF、VEGF+bFGF+SDF+IGF)对EPC细胞增殖及迁徙功能的影响;采用细胞形态观察、双荧光染色法及免疫细胞化学染色方法对培养的EPC进行鉴定。结果猪骨髓EPC以1×104/cm2密度接种在盛有M199,并添加10%FBS和VEGF+bFGF+SDF+IGF细胞因子的培养液时,细胞的增殖能力和迁徙率最高。每组细胞经结合Dil标记的乙酰化低密度脂蛋白(Dil-ac-LDL)和FITC标记的荆豆凝集素(FITC-UEA-1)双色荧光染色鉴定双阳性率>76%,免疫细胞化学检测CD133、CD34、KDR均为阳性。结论通过优化猪骨髓来源EPC的体外培养条件,可使细胞数量增多及功能增强,为后续研究奠定了基础。
Objective To optimize the in vitro culture conditions of porcine bone marrow-derived endothelial progenitor cells (EPCs), and lay the foundation for further study. Methods Bone marrow was extracted from pig iliac bone, mononuclear cells (MNCs) were isolated by density gradient centrifugation and cultured in vitro. Under the same condition of other culture conditions, different cell seeding density (2 × 103 / cm2, 5 × 103 / cm2, 1 × 104 / cm2, 2 × 104 / cm2) (DMEM), different concentrations of FBS (5%, 10%, 20%, 30%) and different concentrations of VEGF (basic fibroblast growth factor) (SDF, IGF and EGF) (VEGF + bFGF, VEGF + SDF, VEGF + bFGF + SDF, VEGF + bFGF + IGF + EGF, VEGF + bFGF + SDF + IGF ) On the proliferation and migration of EPCs. EPCs were identified by cell morphology observation, double fluorescence staining and immunocytochemical staining. Results Porcine EPCs at a density of 1 × 104 / cm2 had the highest proliferative ability and migration rate in M199 medium supplemented with 10% FBS and VEGF + bFGF + SDF + IGF cytokines. Each group of cells was identified by Dil-labeled acetylated low-density lipoprotein (Dil-ac-LDL) and FITC-labeled ditericin agglutinin (FITC-UEA-1) two-color fluorescence staining double positive rate of> 76%, immunocytochemistry Detection of CD133, CD34, KDR were positive. Conclusion Optimizing the culture conditions of porcine bone marrow-derived EPCs can increase the number of cells and enhance their function, which lays the foundation for further research.