小鼠骨髓间充质干细胞体外扩增和分化为神经元样细胞的实验研究

Mouse bone marrow mesenchymal stem cells expansion and differentiate into neuron-like cells in vitro

目的体外分离和扩增小鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs),并观察不同方法诱导BMSCs向神经元样细胞分化的差异,以寻找一种效果理想的BMSCs体外诱导方法。方法取昆明小鼠双股骨骨髓,采用全骨髓贴壁法体外纯化扩增BMSCs,流式细胞术检测其表面标志物,并对其进行成脂、成骨诱导鉴定。分别用化学诱导法和共培养法诱导BMSCs分化为神经元样细胞,比较两种方法所获得的神经元样细胞的细胞形态。结果采用全骨髓贴壁法能有效分离小鼠BMSCs,随着传代次数的增加能得到逐渐纯化的BMSCs,而过多的传代次数则出现细胞生长速度缓慢、核固缩、脱离等衰老征象。流式细胞术检测结果显示P4代BMSCs阳性表达CD29和Sca-1,而阴性表达CD11b。具有成脂、成骨诱导分化的能力。共培养法诱导第5天可见神经细胞样形态且细胞突起数量较多并有分支;化学诱导法培养第7天细胞出现较长突起,形成神经样结构。结论采用全骨髓贴壁法能有效分离纯化小鼠BMSCs,通过共培养法诱导BMSCs分化为神经元样细胞效果优于化学诱导法。

Objective To observe the mouse bone marrow mesenchymal stem cells （ BMSCs） expansion in vitro ,and to find a suitable and practical method of inducing BMSCs into neuron-like cells in vitro by comparing chemical induction and co-culture.Methods BMSCs which that were isolated from mice by the all bone marrow adherence method were cul -tured in the optimum culture condition ,and light microscopy and flow cytometry were used to study the morphology and bio-logical characteristics of BMSCs .The potential to differentiate into osteoblasts and adipocytes was confirmed also .BMSCs were cultured by chemical induction and co-culture methods ,respectively .Cell morphology were compared .Results The all bone marrow adherence method had effective separation of BMSCs ,and they lost their differentiation ability gradually during successive generations .The flow cytometry showed that the CD 29 and Sca-1 of the P4 BMSCs were positive expression ,and CD11b were negative expression .The BMSCs did have the potential to differentiate into osteoblasts and adipocytes in vitro . Neuron-like cells with a larger number of processes were observed on the 5th day in co-culture group while cells with typical nerve cell structure and processes were found on the 7th day in chemical induction group .Conclusions The all bone mar-row adherence method has effective separation of BMSCs .The differentiation effect is better in co-culture group compared with chemical induction group .