大鼠骨髓源性内皮祖细胞的分离培养及鉴定  

Isolation,culture and identification of rat bone marrow derived endothelial progenitor cells

作　　者：陆耀良[1] 康涛[1] 李晓强[2] 韩松[1] 

机构地区：[1]苏州大学附属太仓医院血管外科,江苏苏州215400 [2]苏州大学附属第二医院血管外科,江苏苏州215004

出　　处：《江苏大学学报（医学版）》2013年第6期465-469,共5页Journal of Jiangsu University：Medicine Edition

基　　金：国家自然科学基金资助项目(30972941)

摘　　要：目的:探讨内皮祖细胞(endothelial progenitor cells,EPCs)分离培养鉴定方法及其生物学特性。方法:采用梯密度离心法结合差速贴壁法分离获取大鼠骨髓单核细胞,培养于EGM-2-MV-SingleQuots内皮细胞培养液中,分别取48 h内贴壁细胞(早期EPCs)和48 h后贴壁细胞(晚期EPCs),在倒置显微镜下观察细胞形态,应用流式细胞术鉴定EPCs表面抗原标志CD34,CD133和血管内皮生长因子受体-2(VEGFR-2),激光共聚焦检测EPCs吞噬功能,透射电镜观察EPCs内部超微结构。结果:单核细胞接种后呈小圆形,早期EPCs呈长梭形,晚期以纺锤形为主,培养7 d后有明显干细胞集落;增殖至第6代以后,形态开始逐渐接近于内皮细胞,并出现管腔样结构。内皮祖细胞表面标志CD34,CD133和VEGFR-2均呈阳性表达,能吞噬Dil标记的乙酰化低密度脂蛋白并结合FITC标记的凝集素-1。透射电镜可观察到内皮细胞特征性细胞器Weible-Palade小体。结论:梯密度离心法结合差速贴壁法能够成功地从骨髓中分离、纯化、培养出内皮祖细胞,其增殖能力强,数量充足,生物学特性稳定。Objective:To explore the culture,differentiation and identification method and biological characteristics of the rat bone marrow derived endothelial progenitor cells(EPCs).Methods:We separated the rat bone marrow mononuclear cells using density gradient centrifugation combined with differential sidewall separation,cultured endothelial cell with EGM-2,obtained adherent cells within 48 h(early EPCs)and adherent cells after 48 h(late EPCs),respectively.The cell morphological images were observed under inverted microscope;EPCs surface antigen markers,CD34,CD133 and VEGFR-2 were identified by the flow cytometry,the phagocytosis of EPCs were detected by laser scanning confocal microscope,the internal ultrastructure of EPCs were observed by transmission electron microscope.Results:Mononuclear cells were small round after the first incubation,the late EPCs was given priority to spindle-shaped,and there was obvious stem cell colony after culture in 7 days;till the 6th generations of EPCs,the cellular morphology was gradually close to the endothelial cells,appeared the tube cavity structure.CD34,CD133,VEGFR-2 expressed positive,could absorb Dil-ac-LDL combined with FITC-UEA-1.There was characteristic organelles Weible-Palade(W-P) small body in the EPCs under inverted microscope.Conclusion:The EPC was successfully isolated from bone marrow,and purified,cultured by density gradient centrifugation combined with differential adherent method;it had strong proliferation ability,abundant cell count and stable biological characteristics.

关 键 词：内皮祖细胞 骨髓 干细胞 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]