人骨髓间充质干细胞定向诱导分化为成骨细胞及其鉴定  

INDUCING HUMAN MARROW MESENCHYMAL STEM CELLS INTO OSTEOBLASTS DIRECTIONALLY AND IDENTIFICATION OF THEIR OSTEOGENESIS CHARACTERISTICS

作　　者：殷晓雪[1] 陈仲强[1] 郭昭庆[1] 马庆军[1] 党耕町[1] 

机构地区：[1]北京大学第三医院骨科,北京100083

出　　处：《中国修复重建外科杂志》2004年第2期88-91,共4页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目 (30 2 71 30 5)~~

摘　　要：目的　探讨将成人骨髓间充质干细胞 (MSCs)定向诱导分化为成骨细胞的方法 ,并对所诱导细胞的成骨特性进行鉴定。　方法　分离人骨髓 ,梯度离心并结合全骨髓法进行培养 ,培养基中添加成骨诱导剂地塞米松、β-甘油磷酸钠及抗坏血酸。贴壁细胞传代 ,取第 3代细胞鉴定其成骨特性 ;在倒置相差显微镜下观察细胞形态 ,钙 -钴法染色检测碱性磷酸酶 (AL P)表达 ,免疫组织化学检测 型胶原表达 ,原位杂交检测骨连接素 (ON)、骨桥素 (OP)表达 ,Von Kos-sa染色检测钙结节形成。　结果　第 3代人 MSCs呈典型的成骨细胞形态 ,可连续传代 10次 ;AL P染色阳性率达 85 % ; 型胶原、ON和 OP表达阳性 ;Von Kossa染色可见钙结节形成。　结论　成功地将人 MSCs诱导分化为成骨细胞 ,所诱导的细胞具有典型的成骨细胞特性。Objective To study the method of inducing human marrow mesenchymal stem cells (MSCs) into osteoblasts directionally and to identify osteogenesis characteristics. Methods MSCs were isolated from adult marrow using density gradient separation method and were cultured in conditioned medium containing Dex 10 -8 mol/L,β-GP 10 mmol/L,and AA 50 μg/ml. The MSCs attachment formed soon and passage 3 cells were chosen to check osteogenesis characteristics, including alkaline phosphatase assay with modified calcium-cobalt staining method, type Ⅰ collagen assay with immunohistochemistry, osteopontin and osteonectin assay with in situ hybridization and calcium nodes assay with Von Kossa staining. Results Passage 3 MSCs had typical appearance of osteoblasts and could be passaged continuously till passage 10. The rate of ALP expression was 85%. The expressions of collagen type Ⅰ, osteopontin and osteonectin were positive and calcium nodes were seen by Von Kossa staining. Conclusion We have successfully induced human MSCs into osteoblasts; the induced cells have typical osteogenesis characteristics.

关 键 词：人骨髓间充质干细胞 成骨诱导 成骨分化 成骨细胞 细胞鉴定 组织工程 成骨特性 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学] R318[医药卫生—基础医学]