大鼠骨髓基质干细胞的分离培养与向神经元样细胞诱导分化的实验研究

EXPERIMENTAL STUDY ON ISOLATION, CULTIVATION AND INDUCED DIFFERENTIATION OF BONE MARROW STROMAL STEM CELLS

目的 探讨大鼠骨髓基质干细胞的提取、分离培养和体外扩增的最佳条件 ,研究其在体外培养中定向诱导分化为神经元样细胞的可能。方法 通过密度梯度离心和贴壁培养法从成年大鼠骨髓中分离骨髓基质干细胞 ,进行培养扩增 ,观察其生长特性 ;用 2 巯基乙醇 ( β mercaptoethanol,β ME)对传代细胞诱导分化 ,并通过免疫细胞化学染色鉴定分化细胞的类型。结果 原代培养时形成由基质干细胞组成的细胞集落 ,细胞集落 14d时接近融合 ,传代后 ,细胞体积变大 ,约 5～ 7d传代一次。β ME诱导后 ,70 %以上的细胞在形态上呈神经元样 ,免疫细胞化学染色呈NSE阳性 ,GFAP阴性 ,说明诱导分化的细胞为神经元 ,而不是星形胶质细胞。结论 骨髓基质干细胞在体外培养条件下生长良好 ,并可连续传代 ;在 β

Objective To establish a proper system for the isolation and cultivation of rat bone marrow stromal stem cells (BMSCs), and study the possibility of inducing their differentiation into neuron-like cells in vitro. Method BMSCs were isolated from adult rats using gradient centrifugation and anchoring culture. The proliferative characteristics were observed in primary and passage cultures. After differentiation induced by β-mercaptoethanol (β-ME), immunocytochemistry was used to identify the induced cells. Result Colonies of stromal stem cells were formed in the primary culture and contacted one another on the day 14. In the passage culture, the cells became bigger than those in the primary culture and could be subcultured one generation in 5～7 days. After β-ME induction, about 70% of the BMSCs became neuron-like cells in shape and were positive in NSE but negative in GFAP staining. Conclusion BMSCs can grow well in vitro and be passaged continuously. They can be induced to differentiate into neuron-like cells by β-ME.