双向诱导的骨髓基质干细胞体外成骨与成血管的实验研究

Osteogenesis and angiogenesis in vitro by bidirectional induced bone marrow mesenchymal stem cells in rabbits

目的探讨双向诱导的骨髓基质干细胞（BMSCs）形成内皮细胞、成骨细胞与BMSCs共存体系的体外成骨与成血管的能力。方法采用密度梯度离心法分离培养兔BMSCs。贴壁细胞分为四组：A组（单纯的BMSCs组）、B组（成骨诱导的BMSCs组）、C组（内皮诱导的BMSCs组）和D组（双向诱导的BMSCs组）。通过倒置相差显微镜观察细胞形态学变化，并采用流式细胞仪检测诱导前后的细胞表面抗原，采用茜素红染色观察钙结节，基质胶成血管法观察四组细胞体外成血管的能力。结果①经流式细胞学检测，分离培养的BMSCs主要表达CD90、CD105和CD44；BMSCs向内皮细胞诱导1周后，细胞表面抗原CD34和CD133的表达升高，而CD90和CD105的表达减少，相比诱导前差异有统计学意义（P〈0．05）；BMSCs继续向成骨细胞诱导1周后，细胞表面抗原CD44和HLA-DR升高，相比诱导前差异有统计学意义（P〈0．05）。②茜素红染色结果显示D组的钙结节大于B组，而A组和C组未见钙结节形成。③体外血管新生试验中，C组的管道数目与面积大于D组，差异有统计学意义（P〈0．05），而A组与B组未见管道形成。结论BMSCs双向诱导形成内皮细胞、成骨细胞与BMSCs共存体系，在体外共培养过程中不仅可以形成钙结节，而且可以形成微血管，有利于骨组织和血管联合构建，是一种良好的构建组织工程骨的种子细胞。

Objective To study the potentials of osteogenesis and angiogenesis in vitro of the co-culture system, including endothelial cells, osteoblasts and bone marrow mesenchymal stem cells （BMSCs）, by bidirectional induction. Methods BMSCs were isolated from bone marrow by density gradient centrifugation. The adhesive cells were preserved to passage in culture. The cells were divided into 4 groups： group A （pure BMSCs）, group B （osteogenic induced BMSCs）, group C （endothelial cells induced BMSCs） and group D （bidirectionally induced BMSCs） . The morphological changes were observed through inverted microscopy during induction. Before and after induction, the expressions of cell surface antigens were tested by flow cytometry. The cells in 4 groups were stained by Alizarin Red to observe calcium nodules. The angiogenesis in vitro by the cells in 4 groups were observed on Matrigel. Results Before induction, CD90, CD105 and CD44 were mainly expressed on BMSCs. After BMSCs were induced in vitro into endothelial cells for 1 week, the expressions of cell surface antigens, CD90 and CD105, reduced significantly and those of CD34 and CD133 rose significantly （ P 〈 0.05） . After BMSCs continued to be induced into osteoblasts 1 week later, the flow cytometry showed CD44 and HLA-DR were significantly higher than before （ P 〈 0.05）. No calcium nodules could be observed in groups A and C. The calcium nodules in group D were larger than in group B. In vitro angiogenesis assay revealed that the amount and area of tubes in group B were significantly higher than in group D （ P 〈 0. 05 ）. No tubes were observed in groups A and B. Conclusions Calcium nodules and vessels can be constructed by the co-culture system of endothelial cells, osteoblasts and BMSCs in vitro through bidirectional induction of BMSCs. Since the bidirectionally induced BMSCs can facilitate combined osteogenesis and angiogenesis in vitro, they may be an ideal kind of seed cells for construction of tissue engineered bone.