兔骨髓间充质干细胞的分离培养与鉴定

›› 2015, Vol. 35 ›› Issue (7): 532-537.

兔骨髓间充质干细胞的分离培养与鉴定

王小禹¹,罗红霞²,郭冬梅²,郭晓川²,李鹏³

1. 四川省江油市九○三医院
2. 九○三医院病理科
3. 四川大学华西口腔医学院

收稿日期:2014-11-13 修回日期:2014-12-14 出版日期:2015-07-28 发布日期:2015-08-04
通讯作者: 李鹏 E-mail:lipengfly11@163.com

Isolation and identification of the rabbit mesenchymal stem cells

Received:2014-11-13 Revised:2014-12-14 Online:2015-07-28 Published:2015-08-04

摘要/Abstract

摘要： ［摘要］目的寻求一种安全高效的兔骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs )的体外分离培养和鉴定方法。方法采集兔胫骨骨髓组织，采用全骨髓贴壁培养法体外分离培养和扩增BMSCs；倒置相差显微镜观察细胞，取生长良好的第5代细胞成骨，免疫细胞化学染色和流式细胞术检测细胞表面标志物。结果原代和传代的 BMSCs 贴壁生长，呈漩涡状排列生长；诱导后碱性磷酸酶活性持续高，有钙结节形成并茜素红染色阳性；CD29、CD44、CD105分子呈阳性， CD45、C-kit、CD34分子呈阴性。结论成功建立了兔BMSCs 体外分离纯化、培养的有效方法，扩增的 BMSCs 具有多向分化潜能，是理想的组织工程种子细胞。

关键词: 骨髓间充质干细胞, 流式细胞术, 组织工程

Abstract: Abstract: Objective To explore a safe and efficient method for isolation and identification of the rabbit bone marrow mesenchymal stem cells (BMSCs) in vitro,and to provide an experimental basis for the clinical application of BMSCs.Methods Bone marrow was aspirated from rabbit tibias,and the BMSCs were isolated and cultivated by total adherence method.The cells were observed by inverted phase contrast microscopes.The P5 cells were also induced to differentiate towards osteoblasts and chondroblasts,which were identified by corresponding histochemistry staining and FCM (flow cytometry).Results The primary and passage cells were spindleshaped and grew in colonies.ALP (alkaline phosphatase) activity increased continuously and the calcium nodule which was positive by Alizarin red staining was formed.The surface molecules including CD90,CD44 and CD105 were positive,and the cell surface markers including CD45,CD34 and Ckit were negative.Conclusions The adherence method is a simple and reliable method for rabbit BMSCs cultivation.BMSCs may be ideal seed cells for tissue engineering,which possess power of multidirectional differentiation.

Key words: mesenchymal stem cells, flow cytometry, tissue engineering.

中图分类号:

R329.29

王小禹罗红霞郭冬梅郭晓川李鹏. 兔骨髓间充质干细胞的分离培养与鉴定[J]. 口腔医学, 2015, 35(7): 532-537.

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