Gallo V, Bertolotto A
Section of Neurobiology, Istituto Superiore di Sanita', Rome, Italy.
Exp Cell Res. 1990 Apr;187(2):211-23. doi: 10.1016/0014-4827(90)90084-n.
We have studied the extracellular matrix composition of cultured glial cells by immunocytochemistry with different monoclonal and polyclonal antibodies. Double immunofluorescence experiments and metabolic labeling with [3H]glucosamine performed in different types of cerebellar and cortical cultures showed that bipotential progenitors for type-2 astrocytes and for oligodendrocytes (recognized by the monoclonal antibody LB1 at early stages of their development) synthesize chondroitin sulfate (CS) and deposit this proteoglycan in their extracellular matrix. The distribution of the various [3H]glucosamine-labeled glycosaminoglycans between the intracellular and the extracellular space was different. CS was present both within the cells and in the culture medium, although in different amounts. Bi-potential progenitors became also O4-positive during their development in vitro. At the stage of O4-positivity they were still stained with antibodies against CS. However, when the progenitor cells were maintained in serum-free medium and differentiated into Gal-C-positive oligodendrocytes, they became CS-negative. In the presence of fetal calf serum in the culture medium, the bipotential progenitors differentiated into GFAP-positive type-2 astrocytes. These cells still expressed CS: their Golgi area and their surface were stained with anti-CS antibodies. Staining with monoclonal antibodies specific for different types of CS (4-sulfate, 6-sulfate, and unsulfated) revealed that both bipotential progenitors and type-2 astrocytes synthesized only chondroitin 4-sulfate. Type-1 astrocytes were negative for both the polyclonal and the monoclonal anti-CS antibodies. Finally, type-2 astrocytes and their progenitors were weakly stained with anti-laminin antibodies and unstained with anti-fibronectin. Type-1 astrocytes were positive for both anti-laminin and anti-fibronectin antibodies and appeared to secrete fibronectin in the extracellular space.
我们通过使用不同的单克隆和多克隆抗体进行免疫细胞化学研究了培养的神经胶质细胞的细胞外基质组成。在不同类型的小脑和皮质培养物中进行的双重免疫荧光实验以及用[3H]葡糖胺进行的代谢标记表明，2型星形胶质细胞和少突胶质细胞的双能祖细胞（在其发育早期被单克隆抗体LB1识别）合成硫酸软骨素（CS）并将这种蛋白聚糖沉积在其细胞外基质中。各种[3H]葡糖胺标记的糖胺聚糖在细胞内和细胞外空间的分布是不同的。CS既存在于细胞内，也存在于培养基中，尽管数量不同。双能祖细胞在体外发育过程中也变成O4阳性。在O4阳性阶段，它们仍然被抗CS抗体染色。然而，当祖细胞在无血清培养基中培养并分化为Gal-C阳性少突胶质细胞时，它们变成CS阴性。在培养基中存在胎牛血清的情况下，双能祖细胞分化为GFAP阳性的2型星形胶质细胞。这些细胞仍然表达CS：它们的高尔基体区域和表面被抗CS抗体染色。用针对不同类型CS（4-硫酸酯、6-硫酸酯和未硫酸化）的单克隆抗体染色表明，双能祖细胞和2型星形胶质细胞都只合成硫酸软骨素4-硫酸酯。1型星形胶质细胞对多克隆和单克隆抗CS抗体均为阴性。最后，2型星形胶质细胞及其祖细胞被抗层粘连蛋白抗体弱阳性染色，而被抗纤连蛋白抗体未染色。1型星形胶质细胞对抗层粘连蛋白和抗纤连蛋白抗体均为阳性，并似乎在细胞外空间分泌纤连蛋白。
