SD大鼠骨髓间充质干细胞的分离培养和鉴定

Isolation,Cultivation and Identification of SD Rat Bone Marrow-Derived Mesenchymal Stem Cells

目的:探讨体外分离培养SD大鼠骨髓间充质干细胞rMSCs的方法,检测传代细胞的细胞周期,并分析部分细胞表型,对rMSCs进行初步的鉴定。方法:密度梯度离心结合贴壁培养法分离培养rMSCs,传代扩增,倒置显微镜进行细胞形态学观察,免疫组化检测细胞表面抗原,流式细胞仪检测细胞周期。结果:密度梯度离心结合贴壁培养法能有效分离纯化rMSCs,细胞呈均一的成纤维细胞样,细胞周期显示90.16%P1代细胞处于G0/G1期,免疫组化结果为:CD44、CD29阳性、CD34阴性,说明培养的细胞即非造血干细胞,也非成纤维细胞。结论:本实验分离培养的细胞群与间充质干细胞的生物学特性吻合,说明密度梯度离心结合贴壁培养法能有效分离纯化rMSCs,细胞稳定表达CD44、CD29,是实用、可行的方法,所培养的细胞可用于细胞移植等研究。

Objective：To establish a new method of isolating and culturing rat bone marrow-derived mesenchymal stem cells（rMSCs） in vitro and to analyse cell cycle and their phenotypical properties of the cells after culture expansion as well as rMSCs identified preliminarily.Methods：rMSCs are separated and expanded by gradient centrifugation and adherence culture.Then the morphology of rMSCs are observed by invert microscope and identified by immunohistochemistry method.Cell cycle is detected with flowing cytometry.Resuits： Density gradient centrifugation and plastic adherence method are used for isolation and cultivation of SD rMSCs , rMSCs are fibroblast-like cells,about 90.16% of P1 rMSCs are in G0/G1 phase. The results of immunohistochemistry of rMSCs show that both CD44 and CD29 were positive and CD34, negative, which shows that these cells are no hemopoietic stem cells and fibroblasts. Conclusion： The cells isolated in this test are consistent with MSCs in biological characteristics： gradient centrifugation and plastic adherence methods might be a ideal method for isolation and cultivation of rMSCs, it is feasible MSCs used in cell transplant.