骨髓间充质干细胞建立的成骨细胞培养模型的研究

Establishment and evaluation of rabbit osteoblast cell culture model based on BMSCs

目的:为骨折骨缺损治疗的基础实验研究建立理想的细胞模型,观察兔骨髓间充质干细胞(BMSCs)在体外培养状态下的生长情况,对其向成骨细胞分化及分化能力进行研究.方法:采用全骨髓密度梯度离心法联合差速贴壁法从兔股骨骨髓中提取、分离、纯化BMSCs,取第3代BMSCs细胞分为两组进行培养,A组:为用含有诱导剂的低糖DMEM培养基的诱导培养组;B组:为常规培养组.倒置显微镜及HE染色观察记录各代次细胞形态等特征;CD44,CD90检测细胞表形,通过碱性磷酸酶(ALP)染色及标准化钙结节计数,鉴定细胞成骨活性.结果:兔骨髓间充质干细胞生长状态良好,培养7 d后各组细胞形态趋于一致,多呈长梭形,培养2 wk后对两组细胞进行ALP染色,可见A组细胞质内ALP染色阳性反应明显,B组染色显色弱.21 d时A组细胞茜素红染色后钙化结节数量多且较大,B组有少量钙结节形成,但较小.A组标准化钙结节计数显著高于B组差异具有显著统计学意义(P<0.05).结论:通过密度梯度离心法联合差速贴壁法所获得的BMSCs在常规培养或诱导培养时均可表现出成骨潜能,但诱导培养组的BMSCs的ALP活性更强,成骨能力更确切,可为骨组织工程远期实验研究提供理想的细胞模型.

AIM：To establish a cell culture model for bone disease research,investigate rabbit BMSCs＇ growth and differentiation in vitro.METHODS：Bone marrow mesenehymal stem cells were cultured by density gradient centrifugation and attachment,random select two group from the third generation.Group A：induced by low carbohydrates DMEM;Group B：negative group.The growth of rBMSCs was observed inverted-microscopically,CD44,CD90 were detected,also the HE and ALP stains,assess the activity of cell and counter the mineralized nodus.RESULTS：There were no significant difference between each groups after 7 d culture,the cells apparent long fusiform shape,ALP stains shows significant positive in group A compared with group B after 2 weeks.Mineralized nodus counters show significant higher in group A compared with group B in alizarin red stain after 21 d culture（P〈0.05）.CONCLUSION：Co-application of density gradient centrifugation and attachment in BMSCs cuture shows high capacity of ossification and better activity of ALP,which can provide a cell culture model for bone tissue engineering.