胶质细胞成熟因子对培养的中枢大胶质细胞发育和分化的影响

IN VITRO EFFECTS OF GLIA MATURATION FACTOR ON THE DEVELOPMENT AND DIFFERENTIATION OF CENTRAL MACROGLIAL CELLS

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。

Optic nerves were dissected from 7-day-old Sprague-Dawley rats, dissociated with collagenase and trypsin, and cultured on poly-L-lysine coated glass coverslips. Cultures were grown in the B-S medium (Bottenstein and Sato, 1979) containing 0.5% fetal calf serum(FCS). All of the coverslip cultures were divided into 4 groups: i. e. Ⅰ, 'Ⅰ+GMF', Ⅱ, and 'Ⅱ+GMF'. Optic nerve glial cells were cocultured with glioblast monolayers in both group Ⅱ and group 'Ⅱ+GMF'. Glia maturation factor (GMF) was added to the medium in a concentration of 250 ng/ml for group 'Ⅰ+GMF' and group 'Ⅱ+GMF'. No GMF was added to the medium in both group Ⅰ and group Ⅱ. The coverslip cultures were treated with indirect immunofluorescence to identify the cell types of optic nerve. Cells were double-labled with monoclonal gotibody A2B5 and anti-galactocerebroside(GC) monoclonal antibody, or A2B5 and anti-glisl fibrillary acidicprotein (GFAP) antibody. The bipotential glial progenitor cells (A2B5^+, GC^-; or A2B5^+, GFAP^-), mature oligodendrocytes (A2B5^-, GC^+), immature oligodendrocytes(A2B5^+, GC^+), type 1 astrocytes (A2BS^-, GFAP^+) and type 2 astrocytes (A2B5^+, CFAP^+) can be distinguished.After 5 days in culture, the number of cells in group 'Ⅱ+GMF' showed a significant increase (P<0.02) as compared to day O, but the group Ⅰ exhibited a significant decrease in cell number as compared to the other groups respectively. In the B-S/0.5% FCS medium, the bipotential glial progenitor cells rapidly differentiated into oligodendrocytes, most of which were mature form in group Ⅰ and group Ⅱ, and immature form in group 'Ⅰ+GMF' and group 'Ⅱ+GMF'. These results suggest that both GMF and the factor(s) produced by glioblast monolayers can stimulate the proliferation of bipotential glial progenitor cells and result in the production of large number of oligodendrocytes and support the survival of oligodendrocytes, but in which, the factor(s) produced by glioblast monolayers promotes markedly the differentiation and maturation of oligodendrocytes, whereas GMF may inhibit or delay their maturation.