大鼠骨髓间充质干细胞培养方法的比较及细胞鉴定

Comparison of different culture methods for rat bone marrow mesenchymal stem cells and cultured cell identification

目的研究大鼠骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)不同培养方法的差异。方法采用全骨髓法和密度梯度离心法,对SD大鼠股骨、胫骨骨髓进行分离、纯化和扩增,观察2种方法培养的第0～10代的细胞形态,测定每一代细胞的数量,用流式细胞仪检测第5代细胞CD29、CD34、CD44、CD45的表达率。结果 2种培养方法所获得的细胞形态均呈长梭形,呈特征性的漩涡状生长。全骨髓培养法细胞增殖较快。全骨髓培养法获得的细胞表面标志CD29、CD34、CD44、CD45的表达率分别为91.0%、4.3%、87.1%、0.1%;密度梯度离心法获得的细胞表面标志CD29、CD34、CD44、CD45的表达率分别为96.9%、3.6%、89.7%、0.0%。结论 2种方法培养的细胞均符合BMSCs的特性。全骨髓培养法更经济,操作简单易行,更容易获得大量的BMSCs。

Objective To study the differences of different methods to culture rat bone marrow mesenchy-mal stem cells （BMSCs）.To get morphological observation and identification of the BMSCs that was cul-tured and proliferated.Methods Harvested bone marrow from 2 SD rats’femur and tibia.Whole bone marrow culture and density gradient centrifugation culture were used respectively.To observe the mor-phology in different generations of the cells cultured in two different methods.The number of cells in each generation was determined.Cell surface marker such as CD34,CD29,CD44,and CD45 in P5 generation cells were detected by flow cytometry.Results Cells acquired from two culture methods were long fusi-form,showed characteristic swirling growth.Cell’s proliferation was faster in whole bone marrow culture. Expression rates of cell surface markers CD29,CD34,CD44,and CD45 from the cells obtained from whole bone marrow culture were 91%,4.3%,87.1%,and 0.1%.Expression rates of cell surface markers CD29,CD34,CD44,and CD45 from density gradient centrifugation culture were 96.9%,3.6%,89.7%,and 0%.Conclusion The study proves the cells cultured by both methods are in line with characteristics of BM-SCs.The whole bone marrow culture compared with the density gradient centrifugation culture has not only suc-cessfully isolated BMSCs but also more economical and simple to operate and easier to get a lot of BMSCs.