小鼠骨髓间充质干细胞的分离、培养、纯化及鉴定

目的探讨分离、培养、纯化和鉴定小鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs)方法,观察MSCs体外生长特征。方法取小鼠胫骨和股骨,取出骨髓细胞,用1.073g/ml的Percoll分离液梯度离心,培养皿培养、换液除去非贴壁细胞,获得MSCs,通过传代对MSCs进行纯化和扩增培养。进行形态学观察,测定生长曲线,用流式细胞仪检测P3代MSCs细胞周期及表面抗原。结果 新分离的MSCs呈小圆形,形态规整。培养传代后,细胞大小均匀,形态较一致,多为梭形。P1、P2、P3代MSCs生长曲线均呈S型。P3代MSCs 75.27%的细胞处于G0-G1期。P3代MSCs CD44表达阳性,表达率为88.71%,CD34表达阴性。结论利用密度梯度离心法联合贴壁培养法分离纯化骨髓MSCs,在含15%FBS的DMEM-LG培养基中培养MSCs,可获得稳定生长的昆明小鼠MSCs。培养的MSCs细胞系性状稳定,表型稳定均一,适于做进一步研究。

Isolation,cultivation,purification and identification of mice bone marrow mesenchymal stem cells in vitro

Objective To explore a new method for the isolation, cultivaton, purification and identification of MSCs and observe the biological features of mice MSCs in vitro. Methods Bone marrow was extracted from the tibia and thighbone of mice. The marrow liquid were isolated with 1. 073 g/ml oercoll. MSCs were obtained by removing the nonadherent cells. Then the MSCs were purified and expanded through passage in time. The growth curve was drawn and the. morphology was observed. Cell cycle and the antigen expression of P3 MSCs were measured with FACS. Results The MSCs exhibited a small round shape after fresh separation. After cultivated and passaged,the MSCs were homoge nenously fusiform shaped. The growth curves of P1 ,P2 and P3 MSCs were ＂S＂ shape. The cells of G0- G1 stage account for 75.27% . The expression of CD 44 was positive,while the expression of CD34 was negative. Conclusion The meth od of density gradient centrifugation combined with adherent culture could isolate MSCs from bone marrow simplely. DMEM-LG medium supplemented with 15%fetal bovine serum is suitable for the culture of MSCs. The cultured MSCs lineage is stable and can be used for further research.