文章摘要
蒋晶晶,武晓泓,刘翠萍,徐宽枫,茅晓东,刘超.小鼠骨髓间充质干细胞和内皮祖细胞的分离?培养及鉴定[J].南京医科大学学报,2007,(7):694~697
小鼠骨髓间充质干细胞和内皮祖细胞的分离?培养及鉴定
Isolation, culture and identification of mesenchymal stem cells and endothelial progenitor cells from murine bone marrow
投稿时间:2006-12-20  
DOI:10.7655
中文关键词: 骨髓  间充质干细胞  内皮祖细胞  细胞培养
英文关键词: bone marrow  mesenchymal stem cell  endothelial progenitor cell  cell culture
基金项目:
作者单位
蒋晶晶 南京医科大学第一附属医院内分泌科,江苏 南京〓210029 
武晓泓  
刘翠萍  
徐宽枫  
茅晓东  
刘超  
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中文摘要:
      目的:建立一种高效,稳定的从小鼠骨髓中同时分离培养间充质干细胞(MSC)和内皮祖细胞(EPC)的方法?方法:从小鼠骨髓分离单个核细胞,经差速贴壁结合专用血清或特殊培养基分别扩增MSC和EPC?以成骨?成脂诱导分化鉴定MSC,并以流式细胞术(FCM)检测MSC纯度;以Dil-ac-LDL?FITC-UEA-1荧光双标,结合vWF?CD31免疫组化染色鉴定EPC,并计算其纯度?结果:早期贴壁细胞48 h换液时即可见明显集落形成,1周后即达80%融合,传至第3代后经诱导能够向成骨细胞和脂肪细胞分化,FCM检测CD29?CD34?CD45?CD90阳性率分别为(93.86 ± 1.12)%,(0.48 ± 0.38)%,(1.89 ± 1.49)%,(94.11 ± 3.32)%;2次贴壁细胞经EGM-2 MV专用培养基培养后,第3天开始伸展,第5天可见集落形成,约2周左右可融合近80%,传代后Dil-ac-LDL?FITC-UEA-1双荧光染色阳性率(75.2 ± 4.5)%,vWF?CD31免疫组化染色阳性率分别为(55.7 ± 4.7)%和(52.5 ± 3.6)%?结论:采用该方法可以同时培养扩增骨髓间充质干细胞和内皮祖细胞,效率高,稳定性和重复性好?
英文摘要:
      Objective:To establish an efficient and stable method for simultaneous isolation and culture of murine mesenchymal stem cells and endothelial progenitor cells from bone marrow. Methods:The mononuclear cells were isolated from murine bone marrow and cultured for 2 days. Then the adherent cells were cultured in LG-DMEM with 10% preselected FBS for MSC and the suspending cells were cultured in EGM-2 MV for EPC, respectively. Osteogenic and adipogenic induction was performed on MSCs. The percentage of MSCs was analyzed by flow cytometry(FCM). EPCs were stained with Dil-ac-LDL and FITC-UEA-1, and the expression of vWF and CD31 was also assessed by immunohistochemistry. Results:The early attached cells exhibited clonal morphology as early as 48 hours, which became 80% confluent within 1 week. MSCs could differentiate into osteocytes and adipocytes after induction. The expression of CD29,CD34,CD45 and CD90 were(93.86 ± 1.12)%,(0.48 ± 0.38)%,(1.89 ± 1.49)%,(94.11 ± 3.32)%, respectively. Cells cultured in EGM-2 MV became confluent after 2 weeks,(75.2 ± 4.5)% of which were double positive for Dil-ac-LDL and FITC-UEA-1 staining. The percentages of vWF+ and CD31+ cells were (55.7 ± 4.7)% and (52.5 ± 3.6)%, respectively. Conclusion:An efficient, stable and replicable method for simultaneous isolation and culture of MSCs and EPCs from a single mouse was established.
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