大鼠骨髓源性内皮祖细胞的分离培养及鉴定_陆耀良

第23卷第6期2013年11月江苏大学学报（医学版）

Journal of Jiangsu University （Medicine Edition ）Vol．23No．6Nov．2013

大鼠骨髓源性内皮祖细胞的分离培养及鉴定

陆耀良1，康涛1，李晓强2，韩松

1

（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）

［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48h 内贴壁细胞（早期EPCs ）和48h 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7d 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。［关键词］内皮祖细胞；骨髓；干细胞［中图分类号］Ｒ329．2

［文献标志码］A

［文章编号］1671－7783（2013）06－0465－05

［基金项目］国家自然科学基金资助项目（30972941）［作者简介］陆耀良（1968—），男，江苏太仓人，副主任医师，硕士，主要从事血管外科基础与临床研究；李晓强（通讯作者），教授，博士生

导师

，E-mail ：bright612@163．com Isolation ，culture and identification of rat bone marrow

derived endothelial progenitor cells

LU Yao-liang 1，KANG Tao 1，LI Xiao-qiang 2，HAN Song 1

（1．Department of Vascular Surgery ，the Affiliated Taicang Hospital of Soochow University ，Suzhou Jiangsu 215400；2．Department of Vascular Surgery ，the Second Affiliated Hospital of Soochow University ，Suzhou Jiangsu 215004，China ）

［Abstract ］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 side-wall separation ，cultured endothelial cell with EGM-2，obtained adherent cells within 48h （early EPCs ）and adherent cells after 48h （late EPCs ），respectively．The cell morphological images were observed under inverted microscope ；EPCs surface antigen markers ，CD34，CD133and VEGFＲ-2were 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 ob-vious stem cell colony after culture in 7days ；till the 6th generations of EPCs ，the cellular morphology was gradually close to the endothelial cells ，appeared the tube cavity structure．CD34，CD133，VEGFＲ-2ex-pressed 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 suc-cessfully 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．

［Key words ］endothelial progenitor cells ；bone marrow ；stem cells

DOI:10.13312/j.issn.1671-7783.2013.06.003