犬骨髓间充质干细胞的体外分离培养及鉴定

Isolation,cultivation and identification of canine bone marrow mesenchymal stem cells (BMSCs) in vitro

为了体外高效快捷地分离培养犬(canine)骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs),实验分别用全骨髓差速贴壁法和密度梯度离心法对犬BMSCs进行分离培养,利用免疫组化和流式细胞术检测获得细胞的表面标志抗体,并用成骨和成脂方向的诱导分化等方法对其进行鉴定。结果表明,全骨髓差速贴壁法和密度梯度离心法都能成功培养出犬BMSCs,但后者相对前者获得的细胞经培养后其原代细胞分布更均匀,原代培养达到传代所需的时间更短,成活率更高;两种方法获得的P3和P8细胞的生长曲线基本保持一致;免疫组化和流式细胞术检测犬BMSCs表达CD105、CD90和CD29,但是不表达CD34和CD31;且能成功诱导为成骨细胞和脂肪细胞。证明采用全骨髓差速贴壁法和密度梯度离心法均能够成功分离和培养犬的BMSCs,而密度梯度离心法是一种较全骨髓差速贴壁法更适合犬BMSCs的分离方法。

In order to explore which way can be more efficient and faster to isolate,culture and identifiy the canine bone marrow mesenchymal stem cells(BMSCs)in vitro,the BMSCs were isolated in vitro by whole bone marrow differential velocity adherent and density gradient centrifugation.Immunohistochemistry and flow cytometry were used to examine the surface markers of the cells,and induced their differentiation into osteoblasts and adipocytes.The results showed that canine BMSCs were successfully cultivated by whole bone marrow differential velocity adherent and density gradient centrifugation.Compared with the former,primary cells from the latter method was more uniform after cultivation,and cost shorter time for primary cells proliferation into full confluency along with higher survival rate.The growth curves of undifferentiated cells in P3and P8were similar from the both methods.Immunohistochemistry and flow cytometry showed that CD105,CD90and CD29were expressed in the cells and could be induced into osteoblasts and adipocytes,respectively,while CD34and CD31were not expressed.These results indicated that canine BMSCs could be isolated and cultivated by the both methods,and the density gradient centrifugation method was better compared to the whole bone marrow differential velocity adherent.