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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。
OBJECTIVE: To explore and identify methods for the simultaneous isolation and culture of mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) from mouse bone marrow. Methods Mice bone marrow cells were separated by modified differential adhesion method. 48h later, the adherent cells were passaged to the third generation within 48h, osteogenic, chondrogenic, adipogenic differentiation induction experiment, flow cytometry (FCM) The surface markers were detected after 48h. The adherent cells were collected after 48h. After 3 passages, the vessels were used for angiogenesis assay. After passage 5, CD31 immunofluorescence staining and FCM were used to detect the surface markers. Results Adherent cells were differentiated into bone, cartilage and adipocytes in the third generation within 48h. The positive rates of Sca-1, CD29, CD45 and CD11b were (98.30 ± 0.75)% and (97.47 ± 1.32)%, respectively 1.87 ± 0.15%, (1.03 ± 0.71)% respectively. Adherent cells formed vascular-like structures on the matrigel at 48 h after passage 3, and positive expression of adherent cell-specific surface antigen (CD31) The positive rates of CD34, CD133 and VEGFR2 were (88.90 ± 1.18)%, (92.73 ± 2.90)% and (87.63 ± 1.79)%, respectively. Conclusion Adopting modified differential adhesion method can separate and culture mouse bone marrow MSCs and EPCs at the same time, and is simple, efficient, stable and repeatable.