Percoll法分离犬骨髓单个核细胞与体外成骨诱导培养的实验研究

The Purification and Osteoinductive Culture of Canine Bone Marrow Mononuclear Cells in Vitro by Using Percoll Density Gradient Centrifugation

目的建立犬骨髓单个核细胞(Bone marrow mononuclear cells,BMNCs)的分离培养、体外扩增和成骨诱导方法,为骨缺损的修复提供理想的骨组织工程种子细胞。方法用1.063g/ml percoll液分离2.5ml Beagle犬骨髓,利用细胞贴壁筛选法进行分离、培养、扩增和成骨诱导。结果采用1.063g/ml percoll液可获得纯度较高的BMNCs,接种细胞生长良好,平均倍增周期为1d,经成骨诱导培养后可定向分化为成骨细胞。结论采用1.063g/ml percoll液能够较好的进行Beagle犬BMNCs的分离培养和体外扩增,分离得到的细胞具备骨髓基质干细胞(Bone marrow mesenchymal stem cells,BMSCs)的特性。

Objective To study and establish the method for isolation, culture, proliferation, osteo induction and identification of canine bone marrow mononuclear cells （BMNCs）. Methods 1.063 g/ml Percoll gradient solution separated 2.5 ml canine bone marrow of Beagle. The BMNCs were isolated, cultured, proliferated, osteo induced and identified by the cultivation and selective differentiation. Results With the density gradient centrifugation method, BMNCs were successfully isolated, and 1.063 g/ml Pereoll medium could obtain more purified monocytes. Bone marrow mesenchymal stem cells（BMSCs） smoothly developed during culture, and only about one day for them to become the double of cell number. The BMSCs have shown the ability to give rise to a differentiated cell type of osteocytes in the osteo inductive circumstances. Conclusion Canine BMNCs of Beagle were isolated, cultured and expanded in vitro through the 1.063 g/ml percoll density gradient centrifugation and adherent cell cultivation. These cells showed the characteristics of BMSCs, with self-replenishing ability and the differentiation potential of ossification.