原代大鼠骨髓来源内皮祖细胞培养方法改进及其生物学特性研究  

An improved protocol for primary culture of rat bone marrow-derived endothelial progenitor cells and their biological characteristics

作　　者：李中轩 石宇杰 李俊峡 陈韵岱 Li Zhongxuan;Shi Yujie;Li Junxia;Chen Yundai(不详;Department of cardiology,Seventh Medical Center of Chinese PLA General Hospital,Beijing 100700,China)

机构地区：[1]中国人民解放军总医院第七医学中心心血管内科,北京100700 [2]中国人民解放军总医院第一医学中心心血管内科,北京100853

出　　处：《中国循证心血管医学杂志》2022年第6期727-732,共6页Chinese Journal of Evidence-Based Cardiovascular Medicine

摘　　要：目的探究一种改良的内皮祖细胞(EPCs)分离培养鉴定方法。除应用传统密度梯度离心法与差速贴壁法分离细胞外,还融合集落挑选法增加所培养EPCs纯度,并观察其生物学特性。方法密度梯度离心法分离出大鼠骨髓单个核细胞,结合差速贴壁法以及改良后新增的集落挑选法,在培养过程中不断纯化EPCs。通过吞噬试验,免疫荧光检测,流式细胞术鉴定,增殖实验,活力检测,迁移试验和体外成管实验多方面鉴定EPCs。结果经过改良方法培养的EPCs早期呈现出纺锤形,晚期出现典型的铺路石样集落。(95.7±3.8)%具有吞噬Dil-acLDL和结合FITC-UEA-I能力。免疫荧光发现vWF阳性表达率(96.8±2.8)%,VE-cadherin阳性表达率(97.1±1.4)%。流式结果显示,一次,两次及三次纯化后CD34+和VEGFR2+细胞比例分别为(73.4±2.8)%,(76.8±3.1)%,(80.1±3.4)%,明显高于未纯化细胞(P<0.01)。1×10^(5)个/ml细胞浓度其增殖和活力最好(P<0.01),并应用于后续实验。所培养细胞具有迁移能力及体外成管能力。结论此方法操作简单,培养出的EPCs纯度高,状态好,功能完整。为修复受损管壁及治疗缺血相关性疾病提供足够数量以及纯度的细胞基础。Objective The present study explored an improved protocol for isolating and identifying endothelial progenitor cells.In addition to using traditional density gradient centrifugation and differential adherence method to separate cells,the colony selection method was used to increase the purity of EPCs and investigate the biological characteristics.Methods Rat bone marrow mononuclear cells were isolated by density gradient centrifugation,and EPCs were continuously purified during culture in combination with differential adherence and newly added colony picking after modification.EPCs were identified by many aspects,such as phagocytosis assay,immunofluorescence assay,flow cytometry identification,proliferation assay,viability assay,migration assay,and in vitro tube formation assay.Results The early EPCs appeared with a spindle shape,and the late EPCs showed a typical characteristic endothelial‘cobblestone’morphology.(95.7±3.8)%EPCs were double-positive for the uptake of Dil-acLDL and bound to FITC-UEA-I.Immunocytochemical staining demonstrated that a proportion of vWF positive cells was(96.8±2.8)%,and VE-cadherin positive cells were(97.1±1.4)%.Flow cytometry results showed that the(73.4±2.8)%,(76.8±3.1)%,(80.1±3.4)%of the purified cells co-expressed VEGFR-2 and CD34 after the first,second,and third time purification,which was significantly higher than non-purified cells(P<0.01).The 1×105 cells/ml had the best proliferation and vitality(P<0.01)applied to the subsequent experiments.The cultured cells can migrate and in vitro tube formation.Conclusion This is an easy method to operate,and the EPCs has high purity,good state and complete function.Provided a sufficient source of EPCs for treating ischemic related disease and repairing damaged vascular wall.

关 键 词：内皮祖细胞 增殖 迁移 生物学特性 

分 类 号：R318[医药卫生—生物医学工程]