改良差时贴壁法分离培养鉴定小鼠骨髓间充质干细胞和内皮前体细胞

发布时间：2018-05-14 12:23

本文选题：间充质干细胞 + 内皮前体细胞　；参考：《解剖学报》2015年02期

【摘要】：目的探索同时从小鼠骨髓分离培养间充质干细胞(MSCs)与内皮前体细胞(EPCs)及对其鉴定的方法。方法小鼠骨髓细胞经改良差时贴壁法分离,以48h为时间点,48h内贴壁细胞传至3代后行成骨、成软骨、成脂分化诱导实验,流式细胞术(FCM)检测其表面标记;48h后收集未贴壁细胞,传至3代后行血管形成实验,传至5代后行CD31免疫荧光细胞染色实验,FCM检测其表面标记。结果第3代48h内贴壁细胞可诱导分化为骨、软骨和脂肪细胞,FCM检测Sca-1、CD29、CD45、CD11b阳性率分别为(98.30±0.75)%,(97.47±1.32)%,(1.87±0.15)%,(1.03±0.71)%;第3代48h后贴壁细胞在基质胶上可形成血管样结构,第5代48h后贴壁细胞特异性表面抗原CD31呈阳性表达,FCM检测CD34、CD133、血管内皮生长因子受体(VEGFR2)阳性率分别为(88.90±1.18)%,(92.73±2.90)%,(87.63±1.79)%。结论采用改良差时贴壁法可同时分离培养扩增小鼠骨髓MSCs和EPCs,且简便高效稳定可重复。
[Abstract]:Objective to explore the method of simultaneous isolation and identification of mesenchymal stem cells (MSCs) and endothelial precursor cells (EPCs) from mouse bone marrow. Methods the bone marrow cells of mice were separated by modified delayed adhesion method. The adherent cells were transferred to the third passage within 48 h for osteogenesis, cartilage formation and adipogenic induction. Flow cytometry (FCM) was used to detect the surface labeling of the cells and collect the unadherent cells after 48 h. Angiogenesis was performed after 3 passages and CD31 immunofluorescence staining was performed on the surface of the cells after 5 passages. Results the adherent cells were induced to differentiate into bone within 48 h of the third generation. The positive rates of Sca-1G CD29 CD45 + CD11b in cartilage and adipocytes were 98.30 卤0.75, 97.47 卤1.32, 1.87 卤0.71 and 1.03 卤0.71, respectively. After 48 h of passage 3, the adherent cells formed vascular-like structure on the matrix glue. 48 h after the fifth passage, the positive expression of specific surface antigen (CD31) of adherent cells was detected by FCM. The positive rates of VEGF receptor VEGFR2 were 88.90 卤1.18 and 92.73 卤2.90 respectively. The positive rates of CD34 and CD133 were 92.73 卤2.90 卤87.63 卤1.79, respectively. Conclusion the modified differential time adherence method can be used to isolate and amplify mouse bone marrow MSCs and EPCs simultaneously, and it is simple, efficient, stable and repeatable.
【作者单位】：石河子大学医学院第一附属医院普外科;石河子大学医学院免疫学教研室;
【基金】：国家自然科学基金资助项目(31271458) 人力资源和社会保障部留学回国人员科技活动资助项目(RSLX201201) 兵团科技援疆资助项目(2014AB047)
【分类号】：R329.2

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