猪骨髓来源内皮祖细胞体外培养条件的优化

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。

Optimization of in vitro culture condition for porcine bone marrow-derived endothelial progenitor cells

Objective To optimize the culture condition for porcine bone marrow-derived endothelial progenitor cells (EPCs), so as to lay a foundation for future study. Methods Bone marrow was collected from porcine ilium (n=5). Mononuclear cells (MNCs) were separated by density centrifugation and were induced to differentiate into EPCs in vitro. The influences of different cell inoculation densities (2×103/cm2, 5×103/cm2, 1×104/cm2, and 2×104/cm2), basic medium (EGM, medium 199, and DMEM), FBS (5%, 10%, 20%, and 30%), and combinations of cytokines (vascular endothelial growth factor [VEGF]+basic fibroblast growth factor [bFGF], VEGF+stromal-derived factor [SDF], VEGF+bFGF+SDF, VEGF+bFGF+insulin-like growth factor [IGF]+epidermal growth factor [EGF], and VEGF+bFGF+SDF+IGF) on the proliferation and migration of EPCs were evaluated. EPCs were identified by morphology observation, fluorescent staining, and immunohistochemical method. Results EPCs with the highest proliferation and migration ability were obtained under following condition: at a density of 1×104/cm2 in M199 medium supplemented with 10% FBS and VEGF+bFGF+SDF+IGF. Furthermore, the percentage of double positive cells stained by Dil-ac-LDL and FITC-UEA-1 was higher than 76%, and these cells were also positively stained for CD133, CD34 and KDR immunohistochemically. Conclusion Optimization of in vitro culture condition of porcine EPCs can increase the cell amount and improve their functions, paving a way for future related studies.