构建糖氧剥夺模型大鼠少突胶质细胞体外培养与分离纯化

Isolation and culture of oligodendrocyte from rat and the establishment of oxygen-glucose deprivation model

目的:对原有少突胶质细胞祖细胞培养与分离纯化方法进行改进,以简单、高效获得少突胶质细胞并建立少突胶质细胞糖氧剥夺(oxygen glucose deprivation,OGD)模型。方法:取SD乳鼠脑皮质,采用两种不同的细胞增殖法和两种不同的分离纯化法分四组培养,分别为细胞因子增殖联合摇床震荡分离纯化法组,B104CM增殖联合摇床震荡分离纯化法组,细胞因子增殖联合EDTA消化机械吹打分离纯化法组,B104CM增殖联合EDTA消化机械吹打分离纯化法组。倒置显微镜下观察神经细胞形态学变化并进行细胞计数。A2B5和髓鞘碱性蛋白鉴定少突胶质细胞并分析其纯度。纯化后的少突胶质细胞祖细胞分化培养3 d后分四组培养,正常组正常培养,低氧组分三组,换无糖DMEM培养基,OGD 1、2、3 h。然后采用MTT比色法检测细胞活力;流式细胞术分析细胞的凋亡率。结果:(1)B104CM增殖联合EDTA消化机械吹打分离纯化法较其他3种培养方法收获的少突胶质细胞祖细胞数量多(P<0.05)。(2)A2B5和MBP特异性标记,细胞纯度可达到95%。(3)MTT结果显示:OGD 1 h少突胶质细胞的细胞活力即降低(P<0.05),随着缺氧时间延长细胞活力呈降低的趋势更加明显(P<0.05)。(4)流式细胞测细胞凋亡的结果显示:OGD 1 h、2 h、3 h,细胞的凋亡率分别为14.43%±0.20%,21.99%±0.42%和44.71%±0.20%,均高于正常对照组(6.86%±0.05%,P<0.05)。结论:(1)B104CM增殖联合EDTA消化机械吹打分离纯化培养法与其他三种方法比较实验操作简单,收获的少突胶质细胞祖细胞数量最多,可用于少突胶质细胞的培养。(2)OGD可致少突胶质细胞损伤,且与OGD持续时间密切相关,实验成功的建立OGD损伤模型。

Objective： To investigate the isolation method and growth condition of oligodendrocyte and establish an approach and establishment OGD model to the research of related diseases. Methods： Mixed glial cells of cortices from neonatal rats were primarily culture by two methods,and cells were purified by two methods. Cells were observed by optical microscope. Cells were identified by immumofluorescence staining with A2B5 and MBP. Cells were divided into four groups,OGD 0 h,1 h,2 h,3 h. The viability of the oligodendrocytes was determined by MTT assay,and the apoptosis of oligodendrocytes were measured by flow cytometry after Annexin V-FITC staining. Results： The number of cells harvested by B104 CM proliferation and EDTA is larger than the other three methods（ P〈0. 05）. A2B5 and MBP-specific markers are positive and the purification is more than 95%. Using MTT,we found that the survival rate in the OGD cells decreased after the setting of oxygen glucose deprivation for 1 hours（ P〈0. 05）,While the damage of oligodendrocytes aggravated gradually in OGD cells following the prolongation of times（ P〈0. 05）. Using flow cytometry assays,we found that the apoptosis rate in the OGD cells model was 14. 43 ± 0. 20%,21. 99 ± 0. 42% and 44. 71 ± 0. 20% after the setting of oxygen glucose deprivation for 1 h,2 h and 3 h as compared with those in control group（ 6. 86 ± 0. 05%,P〈0. 05）. Conclusion： oligodendrocytes were cultured and OGD model was established successfully.