大鼠骨髓来源内皮祖细胞的分离培养及鉴定  

Isolation and Culture of Rat Bone Marrow-Derived Endothelial Progenitor Cells

作　　者：戴小珍[1,2] 王兰[1] 潘克俭[1] 何浪[1] 李红[1] 姜鹤群 

机构地区：[1]成都医学院生物医学系,成都610500 [2]生物流变科学与技术教育部重点实验室(重庆大学),重庆400044 [3]成都医学学院第一附属医院肿瘤科,成都610500

出　　处：《成都医学院学报》2013年第3期242-246,共5页Journal of Chengdu Medical College

基　　金：国家自然科学基金项目(NO:81200917);四川省教育厅资助科研项目(NO:11ZA205);重庆大学"生物流变科学与技术"教育部重点实验室访问学者基金项目(NO:CQKLBST-2012-003)

摘　　要：目的建立体外分离培养及鉴定大鼠骨髓来源内皮祖细胞(endothelial progenitor cells,EPCs)的方法。方法采用密度梯度离心法从大鼠骨髓中分离得到骨髓单个核细胞,并结合差时贴壁法,收集24h后未贴壁细胞,然后用添加有多种细胞因子的内皮细胞基础培养基(endothelial basal medium,EBM-2)诱导培养获取EPCs。通过形态学观察、CD133和VEGFR-2抗体免疫荧光染色并结合FITC-UEA-1和Dil-ac-LDL的摄取功能来对EPCs进行鉴定分析,同时通过体外管样结构形成能力对EPCs进行功能性评价。结果在EBM-2培养基中诱导培养7d后,出现类似于血岛样的细胞集落,集落中央的细胞呈圆形,周边的细胞呈放射状;80%以上的细胞都是CD133+/VEGFR-2+细胞,并且这些细胞同时能够摄取Dil-ac-LDL和结合FITC-UEA-1;EPCs在基质胶表面形成管腔样结构。结论密度梯度离心法结合差时贴壁法从骨髓中分离获得的单个核细胞,然后经EBM-2条件培养基诱导培养可获得较高纯度的EPCs。Objective To develop an efficient method to isolate and culture bone marrow-derived endothelial progenitor ceils （EPCs）. Methods Mononuclear cells （MNCs） were isolated from rat bone marrow by density gradient centrifugation and then plated on 6-well plates. After a 24 h culture, non-adhesion cells were collected,and then seeded on fibroneetin pre-eoated 6-well plates,cultured with EBM-2 supplemented with various growth factors. EPCs were identified by immunofluorescence staining for CD133 and VEGFR-2, combined with the uptaking ability of FITC-UEA-1 and Dil-Ae-LDL. Further functional assessment of EPCs were performed by tube-like structure formation in vitro. Results After a 7 day culture under endothelial-specific conditions,bone marrow-derived MNCs developed a spindle-shape appearance and typical cell clusters. The appearance and organization of these cell dusters resembled as the characteristic blood island-like cell clusters. Immunofluoreseent staining assay demonstrated that most of the cells （up to 80%） were positive for CD133＋ and VEGFR-2＋. And it was found that the ceils were positive for Dil-ac-LDL uptaking and lectin binding. The double positive ceils were identified as differentiating EPCs. Furthermore,EPCs could form tube-like structures on matrix gel. Conclusion The results suggested that high-purity EPCs could be harvested by EBM-2 medium induced-culturing MNCs that were isolated by density gradient centrifugation combining with differential adhersion time methods.

关 键 词：内皮祖细胞 分离培养 鉴定 管样结构 

分 类 号：R394.2[医药卫生—医学遗传学]