Yin Xiao-xue, Chen Zhong-qiang, Guo Zhao-qing
Department of Orthopedic Surgery, Third Hospital of Peking University, Beijing, P. R. China 100083.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2004 Mar;18(2):88-91.
To study the method of inducing human marrow mesenchymal stem cells (MSCs) into osteoblasts directionally and to identify osteogenesis characteristics.
MSCs were isolated from adult marrow using density gradient separation method and were cultured in conditioned medium containing Dex 10(-8) mol/L, beta-GP 10 mmol/L, and AA 50 micrograms/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.
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.
We have successfully induced human MSCs into osteoblasts; the induced cells have typical osteogenesis characteristics.
研究将人骨髓间充质干细胞（MSCs）定向诱导为成骨细胞的方法并鉴定其成骨特性。
采用密度梯度分离法从成人骨髓中分离MSCs，并在含有10⁻⁸mol/L地塞米松（Dex）、10mmol/Lβ-甘油磷酸钠（β-GP）和50μg/ml抗坏血酸（AA）的条件培养基中培养。MSCs很快贴壁，选择第3代细胞检测成骨特性，包括采用改良钙钴染色法进行碱性磷酸酶检测、采用免疫组织化学法进行Ⅰ型胶原检测、采用原位杂交法进行骨桥蛋白和骨连接蛋白检测以及采用Von Kossa染色法进行钙结节检测。
第3代MSCs具有典型的成骨细胞外观，可连续传代至第10代。碱性磷酸酶表达率为85%。Ⅰ型胶原、骨桥蛋白和骨连接蛋白表达呈阳性，Von Kossa染色可见钙结节。
我们已成功将人MSCs诱导为成骨细胞；诱导后的细胞具有典型的成骨特性。
