SD大鼠少突胶质前体细胞的纯化及氧糖剥夺对其活性的影响

Purification and identification of oligodendrocyte precursor cells from rat cortex and the effect of oxygen-glucose deprivation model on its activity

目的:纯化少突胶质前体细胞,并探讨氧糖剥夺不同时间对其活力的影响。方法:取SD乳鼠的脑皮质,采用差速贴壁、振荡分离纯化、限定培养基定向培养法,获取少突胶质前体细胞。镜下观察少突胶质前体细胞分化过程中各阶段细胞的形态变化。采用特异性抗体A2B5、MBP、GFAP、IBA - 1标记纯化后少突胶质前体细胞、成熟少突胶质细胞、星形胶质细胞和小胶质细胞所占比例,以此分析纯化后的细胞纯度。体外制备细胞氧糖剥夺(oxygen glucose deprivation,OGD)模型,采用CCK - 8法观察其对少突胶质细胞系增殖活性的影响。结果:(1)利用针对少突胶质前体细胞、成熟少突胶质细胞、星形胶质细胞和小胶质细胞的特异性抗体A2B5、MBP、GFAP和IGB - 1进行标记,A2B5、MBP染色阳性细胞分别占96.2%、2.1%,GFAP和IBA - 1染色阳性细胞小于3%。(2) MTT检测显示,随着OGD干预时间的延长,细胞活力呈降低的趋势。结论:(1)联合应用差速贴壁、恒温振荡分离和条件限定培养基可以获得高纯度的少突胶质前体细胞;(2)干预 3 h 适宜用于体外少突胶质前体细胞的OGD模型。体外建立的OGD模型可导致少突胶质前体细胞的损伤,使其细胞活性下降。

Objective: To explore the methods of isolation and purification of oligodendrocyte precursor cells (OPCs) in the brain of newborn Sprague- Dawley(SD) rats, and to explore the effects of different intervention times of oxygen glucose deprivation(OGD) on oligodendrocyte viability. Methods: The high purity oligodendrocyte precursor cells were obtained from the cerebral cortex of SD neonatal rats by differential adhesion, oscillatory separation and purification, and restricted medium directional culture method. The morphological changes of oligodendrocyte precursor cells during differentiation were observed under microscope. The purified oligodendrocyte precursor cells, mature oligodendrocytes, astrocytes and microglia were labeled with specific antibodies A2B5, MBP, GFAP and IBA _ 1 to analyze the purity of the purified cells. The effect of OGD on the proliferation of oligodendrocyte cell line was observed by CCK- 8 assay. Results: Specific antibodies A2B5, MBP, GFAP and IGB- 1 were used to label oligodendrocytes, mature oligodendrocytes, astrocytes and microglia. A2B5, MBP positive cells accounted for 96.2%, 2.1%, GFAP and IBA- 1 staining positive cells were less than 3%. The results of MTT detection showed that in OGD model, the cell viability decreased with the prolongation of OGD intervention time. Conclusion:(1) High purity oligodendrocyte precursor cells can be obtained by combination of differential adhesion, constant temperature oscillation separation and conditioned medium.(2) The OGD model established in vitro can cause the damage of oligodendrocyte precursor cells and decrease the activity of oligodendrocyte precursor cells. 3 h intervention is suitable for the establishment of OGD model of oligodendrocyte precursor cells in vitro .