成人间充质干细胞体外诱导分化为成骨细胞的研究

Experimental research on the adult human bone marrow mesenchymai stem cells differentiating into osteoblasts

目的研究成人骨髓间充质干细胞（bone marrow mesenchymal stem cells，BMSCs）体外培养定向诱导分化为成骨细胞，探讨其作为骨组织工程种子细胞的可行性和应用价值。方法抽取健康成年骨髓，Ficoll密度梯度离心结合贴壁培养法经连续传代后改用含10nmol／L地塞米松、10nmol／Lβ-甘油磷酸钠、50μmol/L维生素C的条件培养基培养，在相差显微镜下观察细胞形态变化，流式细胞仪细胞表面分子标志鉴定，免疫组化和RT-PCR检测Ⅰ型胶原蛋白的表达，同时测定细胞内碱性磷酸酶的含量。结果BMSCs原代和传代培养的细胞具有活跃的增殖能力，诱导培养后细胞形态向成骨细胞转化，经RT-PCR、流式细胞仪、细胞碱性磷酸酶活性、糖原染色鉴定为成骨细胞。结论BMSCs经合理的体外诱导培养后符合成骨细胞的形态特征和生物学特性，有望成为理想的骨组织工程种子细胞来源。

Objective To establish an ideal culture method on the differentiation of the adult human bone marrow mesenchymal stem cells （BMSCs） to osteoblasts in vitro. Methods The bone marrows were obtained by aspiration from healthy volunteers. BMSCs were isolated and purified by modified ficoll density gradient centrifugation combined with adherence culture method. MSCs were induced to osteoblasts in vitro in a condition medium in subculture including dexamethasone, vitamin C, and β- glycerophosphate. The morphological changes and growth characteristics were examined by phase-contrast microscopy. Cell surface markers CD34 and CD45 were tested by flow cytometric analyses. Alkaline phosphatase activity and expression of type I collagen were also studied. Results Adult BMSCs presented active proliferation in primary and passage cultures in vitro. Cells were spindleshaped. The cell surface markers CD34 and CD45 were positive before differentiation and were negative after differentiation. Cells were successfully induced into osteobiasts. They were identified through morphological character, specific surface antigens and osteogenic differentiation. Immunochemical expression of type Ⅰ collagens of the differentiated cells revealed the characteristics of osteogenic differentiation with strong staining. Alkaline phosphatase activity were increase. Conclusion The osteoblasts could be induced from human BMSCs, which have typically morphological and biological characteristics of osteoblasts.