Wistar大鼠骨髓间充质干细胞的体外培养及成骨诱导实验

Culture of rats' bone marrow derived mesenchymal stem cells in vitro and ossification induction experiment

目的探讨Wistar大鼠骨髓间充质干细胞(BMSCs)在体外进行分离、纯化以及定向诱导成骨细胞的方法 ,为进一步利用BMSCs移植治疗骨组织缺损奠定基础。方法体外分离培养BMSCs,并进行定向的成骨诱导分化,采用茜素红染色、Ⅰ型胶原免疫组织化学染色和免疫组化col-Ⅱ染色等方法进行鉴定。结果经过体外分离培养的大鼠BMSCs在显微镜下为贴壁生长的成纤维样细胞,其形态呈长梭形。经过第一次传代后的BMSCs形态趋于均一,呈漩涡样或者菊花样生长;传代后在7 d内BMSCs生长迅速,在7 d后细胞密度增加,出现接触抑制现象,而使细胞的生长速度减慢。经定向诱导后BMSCs明显表达为成骨表型。结论 BMSCs是一种易于在体外分离培养和扩增的细胞群,经体外定向成骨诱导后的BMSCs具有典型的成骨细胞的形态和功能性特征,可以作为骨组织工程的种子细胞。

Objective To explore the approach to separate and purify Wistar rats’ bone marrow derived mesenchymal stem cells（BMSCs） in vitro and directionally induce osteoblast in order to provide a foundation for further use of BMSCs transplantation to treat bone defect disease. Methods BMSCs from Wistar rats were isolated and cultured in vitro and directionally induced to osteoblastic differentiation. Identification was made through the methods of alizarin red staining and collagen type Ⅰ and col- Ⅱimmunohistochemical staining. Results The BMSCs separated and cultured in vitro presented long spindle-shaped fibroblast-like cells with adherence growth under the microscope. The morphology of BMSCs after the first passage tended to be homogeneous with swirl or chrysanthemum like growth. Within 7 days after the passage, BMSCs showed rapid growth. Within 7 days after the passage,the cells grew fast, and the cell density increased after 7 days. Contact inhibition occurred which decreased the cell growth velocity.After the directional induction, BMSCs presented apparent osteogenous phenotype. Conclusion BMSCs can be easily isolated and multiplied in vitro. Those cells which were induced to osteoblastic differentiation present typical osteoblast morphology and functional characteristics. Therefore, BMSCs are the ideal seed cells for the bone tissue engineering research.