一种分离培养少突胶质前体细胞的改进方法

Improved method on isolation and culture of rat oligodendrocyte precursor cells

目的:对少突胶质前体细胞的分离培养方法进行改良,为今后细胞移植治疗研究提供基础。方法:在大鼠少突胶质前体细胞分离纯化培养过程中添加不同浓度bFGF(5、10、20 ng/ml),显微镜观察少突胶质前体细胞在体外的生长情况,台盼蓝实验检测细胞存活率和获得率,免疫细胞化学技术进行细胞鉴定,流式细胞术检测细胞纯度。结果:在少突胶质前体细胞培养过程中添加10 ng/ml的bFGF,可明显提高少突胶质前体细胞的产出率和纯度。分离的少突胶质前体细胞呈A2B5、NG2阳性,能进一步分化为成熟少突胶质细胞且表达MBP和O4。结论:在培养中添加适当浓度的bFGF的方法能有效获得高纯度的少突胶质前体细胞。

Objective： To obtain healthier and purer oligodendrocyte precursor cells （OPCs） for the experiment of cell transplantation, the methods was modified in rat OPCs isolation and culture. Methods： Using bFGF of different concentra- tions （ 5, 10, 20 ng/ml） in the culture of OPCs, the growth pattern of OPCs in vitro was investigated by microscopy, cell survival rate and viable cell number were detected using trypan blue assay, cells were further identified with the immuno- fluorescent staining, and the purity of OPCs was detected by Flow cytometry. Results： Adding 10 ng/ml bFGF in OPC culture process, the purity and viable cell number were significantly improved. OPCs were immunoreactive with the spe- cific antibodies A2B5 and NG2. In addition, these OPCs progressively differentiated into mature oligedendrocytes which specifically expressed MBP and 04. Conclusion： The method adding moderate concentration of bFGF in the culture of OPCs, was suitable and effective to obtain OPCs in high purity .