兔骨髓间充质干细胞定向诱导分化为成骨细胞的研究

Study on rabbit bone marrow mesenchymal stem cells differentiation to the osteoblasts in vitro

目的 探讨兔骨髓间充质干细胞﹙bone marrow mesenchymal stem cells, BMSCs﹚体外分离培养、诱导分化为成骨细胞的方法,并研究其生物学特性.方法 采用体外细胞培养技术自兔股骨、胫骨及肱骨中分离BMSCs进行纯化、培养.用地塞米松、β-甘油磷酸钠、维生素C定向诱导BMSCs分化为成骨细胞,改良MTT法测定其生长曲线,NBT/BCIP染色法、P-对硝基苯基质法及茜素红染色法检测BMSCs的成骨能力.结果 成骨诱导1～5 d,培养的细胞增殖旺盛,3～5 d即可融合成单层,随着诱导时间的延长,细胞增殖速度减慢,出现细胞聚集的现象,培养细胞逐渐汇合呈铺路石状,细胞形态变为胞体较小的立方形,继续诱导,可出现多角形成骨样细胞,胞外基质分泌逐渐增多,胶原堆积、钙盐沉积,10～12 d形成不透光矿化结节,ALP活性增高,茜素红染色阳性,表示BMSCs向成骨细胞分化.结论 兔BMSCs经体外分离、诱导培养,可以定向分化为成骨细胞.

Objective To investigate the isolation and cultivation of bone marrow mesenchymal stem cells （ BMSCs ） from the bone marrow of rabbit and induce them to differentiate into osteoblasts in vitro. Methods BMSCs were harvested from the femurs, tibias and humerus bones of the rabbit, then separated, purified and proliferated. After primary culture, the subcuhured cells were cultured in osteogenesis inducing medium including dexamethasone, β-glycerophosphate, L-ascorbic acid, then stained with NBT/BCIP and alizarin red. Results The BMSCs of rabbit showed active proliferative capability in primary and passage cultures. After 8 days of osteoblast inducing, the BMSCs congregated , showed typical cuboidal shape and formed mineralized nodule. The expression of alka- line phosphatase was positive. Conclusion The BMSCs isolated and cultured can be induced into osteogenesis committed differentiation.