外周血早晚期内皮祖细胞的生物学特性及鉴定的初步研究  

BIOLOGICAL FEATURES AND IDENTIFICATION OF ENDOTHELIAL PROGENITOR CELLS FROM PERIPHERAL BLOOD

作　　者：乔威[1] 周敏[2] 刘长建[2] 乔彤[2] 

机构地区：[1]江苏省中医院血管外科,南京210029 [2]南京大学医学院附属鼓楼医院血管外科

出　　处：《中国修复重建外科杂志》2015年第7期870-877,共8页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：江苏省干部保健科研项目(BJ14013)~~

摘　　要：目的通过人外周血分离培养早晚期内皮祖细胞（endothelial progenitor cells,EPCs）,比较两类EPCs的特性,以期找到可靠的稳定生物学表征及鉴定方法鉴定EPCs。方法通过密度梯度离心法分离人外周血单核细胞,体外种植于内皮细胞全培养基诱导培养,分别于培养4~7 d及2~3周获得早、晚期EPCs。对两类EPCs的细胞形态、增殖能力、细胞表型、细胞因子表达情况、体外成血管能力及一氧化氮（nitric oxide,NO）释放能力进行检测比较,并以成熟人主动脉内皮细胞（human aortic endothelial cells,HAECs）作为阳性对照。结果两类EPCs形态不同,早期EPCs形成纺锤样细胞簇,而晚期EPCs呈鹅卵石样外观;晚期EPCs具有高增殖潜能,可在基质胶上形成毛细管状结构,而早期EPCs则不具备该特性;两类EPCs均可摄取乙酰化低密度脂蛋白,并能与荆豆凝集素Ⅰ结合;流式细胞仪检测发现早期EPCs不表达CD34和CD133,但造血干细胞标记CD14和CD45表达却呈阳性,而晚期EPCs对于内皮型表面标记CD31和CD34表现为强阳性,但CD14、CD45、CD133表达则呈阴性。RTPCR分析发现早期EPCs的血管内皮生长因子受体2及血管内皮钙黏蛋白基因的相对表达量显著低于晚期EPCs及HAECs（P〈0.05）,而血管性血友病因子及内皮型一氧化氮合成酶（endothelial nitric oxide synthase,eNOS）基因的相对表达量比较差异无统计学意义（P〉0.05）。细胞因子分泌方面,早期EPCs培养上清液中的VEGF、粒细胞集落刺激因子及IL-8浓度明显高于晚期EPCs（P〈0.05）。Western blot检测示,两类EPCs均表达eNOS,但培养5周的晚期EPCs的eNOS表达水平高于早期EPCs（P〈0.05）;两种EPCs均可释放NO,但NO产量差异无统计学意义（P〉0.05）。结论 eNOS的表达及NO释放能力作为内皮细胞的可靠生物学特征可用于EPCs鉴别,多方法联合方式用于鉴定EPCs更为可行。Objective To compare the biological features of early and late endothelial progenitor cells（EPCs） by isolating and culturing early and late EPCs from the human peripheral blood so as to find some unique properties of EPCs and to propose a suitable strategy for EPCs identification. Methods Mononuclear cells were isolated from the human peripheral blood using density gradient centrifugation. Then, the cells were inoculated in human fibronectin-coated culture flasks and cultured in endothelial cell basal medium 2. After 4-7 days and 2-3 weeks culture, early and late EPCs were obtained respectively. The morphology, proliferation potential, surface markers, cytokine secretion, angiogenic ability, and nitric oxide（NO） release were compared between 2 types of EPCs. Meanwhile, the human aortic endothelial cells（HAECs） were used as positive control. Results The morphology of early and late EPCs was different： early EPCs formed a cell cluster with a spindle shape after 4-7 days of culture, and late EPCs showed a cobblestone appearance. Late EPCs were characterized by high proliferation potential and were able to form capillary tubes on Matrigel, but early EPCs did not have this feature. Both types EPCs could ingest acetylated low density lipoprotein and combine with ulex europaeus I. Flow cytometry analysis showed that early EPCs did not express CD34 and CD133, but expressed the CD14 and CD45 of the hematopoietic stem cell markers; however, late EPCs expressed CD31 and CD34 of the endothelial cell markers, but did not express CD14, CD45, and CD133. By RT-PCR analysis, the expressions of vascular endothelial growth receptor 2 and vascular endothelial cadherin in early EPCs were significantly lower than those in the late EPCs and HAECs（P〈0.05）, but no significant difference was found in the expression of von Willebrand factor and endothelial nitric oxide synthase（eNOS） between 2 type EPCs（P〉0.05）. The concentrations of vascular endothelial growth factor,granulocyte colony-stimulating factor, and

关 键 词：内皮祖细胞 内皮型一氧化氮合成酶 一氧化氮 细胞鉴定 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]