成人骨髓基质干细胞体外诱导成骨细胞

A study of osteogenic induction of adult human bone marrow stromal stem cells in vitro

目的 建立成人骨髓基质干细胞(BMSCs)分离、扩增以及诱导和分化为成骨细胞的体外培养方法,为骨组织工程选择理想的种子细胞来源。方法 抽取健康成人骨髓组织,用 Percoll分离液分离出骨髓中的单个核细胞,在含体积分数为 10%小牛血清的高糖 DMEM培养液中,置于37℃、含体积分数为5%的CO2 湿化空气孵箱中培养,通过传代培养扩增BMSCs,传三代时改用含地塞米松、β 甘油磷酸和维生素C的条件培养基培养,用倒置显微镜、HE染色观察增殖和分化情况,并测定碱性磷酸酶活性和钙结节形成能力。结果 体外培养的成骨细胞生长良好,表现出与典型的成骨细胞相似的形态特征和生物学特性。结论 所建立的成人 BMSCs分离、扩增以及诱导和分化为成骨细胞的体外培养方法稳定和实用,可作为骨组织工程种子细胞来源的常规方法之一。

Objective To establish a method for isolation,expansion and osteogenic induction of adult human bone marrow stromal cells in vitro and to select an ideal source of seed cells for bone tissue engineering.Methods Bone marrow was aspirated from healthy adult human.The mononuclear cell fraction of the marrow was enriched by centrifugation on a percoll cushion and cultured in high-glucose DMEM containing volumes of 10% fetal bovine serum.The passaged cells were incubated at 37℃ in a humidified 50% CO 2 environment.Subsequently,dexamethasone,beta-sodiun glycerophosphate and ascorbic acid were added into culture mediun to feed their passages.Proliferations and differentiations of all the cultured cells were observed continually under inverted microscope,a light microscope with HE stained.Activities of alkaline phosphatase and abilities of formation for calcifying nodules were elicited.Results The human BMSCs cultured survived well and the cultured cells elicited a biological and morphologic characteristics similar to those of osteoblasts. Conclusion The method for isolation and cultivation of hBMSCs is stable and practical,and can be used as a routine of culturing seed cells of bone tissue engineering.