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用胶原酶和胰酶分离7天大鼠视神经胶质细胞,培养在涂有多聚赖氨酸的盖玻片上,培养液用仅含0.5％胎牛血清的B-S培养基(B-S/0.5％FCS,Bottenstein and Sato,1979)。把盖玻片培养物分为Ⅰ、“Ⅰ+GMF”、Ⅱ和“Ⅱ+GMF”4组,Ⅱ和“II+GMF”组分别与单层成胶质细胞联合培养,“Ⅰ+GMF”和“Ⅱ+GMF”组的培养液内添加胶质细胞成熟因子(GMF)250ng/ml,Ⅰ和Ⅱ组的培养液不加GMF。用单克隆抗体A2B5与抗半乳糖脑苷脂(GC)单克隆抗体,或A285与抗胶质原纤维酸性蛋白(GFAP)抗体进行双标记间接免疫荧光染色,以鉴別视神经培养物中的双潜能胶质祖细胞(A2B5~+,GC~-;或A2B5~+,GFAP~-)、成熟少突胶质细胞(A2B5~-,GC~+)、未成熟少突胶质细胞(A2B5~+,GC~+)、1型星形胶质细胞(A2B5~-,GFAP~+)和2型星形胶质细胞(A2B5~+,GFAP~+)。培养第5天,“Ⅱ+GMF”组的细胞数目有显著性增加(P<0.02),Ⅰ组细胞数目比其余3组低。在B-S/0.5％FCS培养基中,各组的双潜能胶质祖细胞均迅速分化为少突胶质细胞,但在Ⅰ和Ⅱ组以成熟少突胶质细胞为主,而在“Ⅰ+GMF”和“Ⅱ+GMF”组则未成熟少突胶质细胞较多。以上实验结果提示:GMF和成胶质细胞分泌的因子有刺激双潜能胶质祖细胞增殖导致生成大量少突胶质细胞及支持其生存的作用。但在此过程中,成胶质细胞分泌的因子明显促进少突胶质细胞分化成熟,而GMF却呈现抑制或延缓其成熟的作用。
The rat glial cells were isolated with collagenase and trypsin for 7 days, cultured on polylysine-coated coverslips, and incubated with BS medium containing 0.5% fetal bovine serum (BS / 0.5% FCS , Bottenstein and Sato, 1979). The coverslip cultures were divided into four groups: Ⅰ, “Ⅰ + GMF”, Ⅱ and “Ⅱ + GMF”. Groups Ⅱ and Ⅱ + GMF were cultured with monolayer of glial cells, And GM-CSF supplemented with 250ng / ml of GMF. The culture medium of groups I and II was without GMF. Dual-labeling indirect immunofluorescent staining with monoclonal antibody A2B5 and anti-galactocerebrosidase (GC) monoclonal antibodies, or A285 and anti-glial fibrillary acidic protein (GFAP) antibodies were used to identify double Potential glial progenitor cells (A2B5 ~ +, GC ~ - or A2B5 ~ +, GFAP ~), mature oligodendrocytes (A2B5 ~ -, GC ~ +), immature oligodendrocytes (A2B5 ~ +, GC ~ +), type 1 astrocytes (A2B5 ~ -, GFAP ~ +) and type 2 astrocytes (A2B5 ~ +, GFAP ~ +). On day 5, the number of cells in “Ⅱ + GMF” group was significantly increased (P <0.02), and the number of cells in group Ⅰ was lower than the other three groups. In BS / 0.5% FCS medium, all the groups of the bi-potential glial progenitor cells differentiated into oligodendrocytes rapidly, but the mature oligodendrocytes were predominant in the groups Ⅰ and Ⅱ, while in the “Ⅰ + GMF ”and“ Ⅱ + GMF ”group immature oligodendrocytes are more. The above experimental results suggest that the factors secreted by GMF and glial cells stimulate the proliferation of double-potential glial progenitor cells and lead to the formation of a large number of oligodendrocytes and support their survival. However, in the process, the factors secreted by glial cells significantly promote the differentiation and maturation of oligodendrocytes, while GMF has the effect of inhibiting or delaying its maturation.