Mochizuki Y, Park M K, Mori T, Ogura A, Kawashima S
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Bunkyo-ku, Japan.
Gerontology. 1998;44(1):1-8. doi: 10.1159/000021975.
We found that neuroblastoma x glioma hybrid NG108-15 cells accumulated lipofuscin-like autofluorescent materials during neuronal differentiation in culture in a medium containing 1% fetal calf serum, 1 mM dibutyryl cyclic AMP and 1 mM theophylline. The emission maximum of the lipofuscin-like autofluorescent materials was between 500 and 550 nm. Granules positive to acid phosphatase and periodic-acid Schiff were increased, as were the autofluorescent granules in NG108-15 cells. Thiolprotease inhibitors, N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucine-4-aminobutyla mide (E-64) and acetyl-Leu-Leu-Arg (leupeptin), markedly accelerated the accumulation of the lipofuscin-like autofluorescent materials in NG108-15 cells. On the other hand, activities of lysosomal thiolproteases, cathepsin B, C and L, were increased during neuronal differentiation. Protein content in the cells was gradually increased with the neuronal differentiation, and the rise was significantly accelerated when proteolysis was inhibited by E-64. These results suggest that the lipofuscin-like autofluorescent materials contain peptidic substances as a component, and indicate that the increase in hydrolytic activities of thiolproteases during neuronal differentiation is not enough for the hydrolysis of peptidic substrates, resulting in the accumulation of autofluorescent materials in NG108-15 cells.
我们发现，在含有1%胎牛血清、1 mM二丁酰环磷腺苷和1 mM茶碱的培养基中培养时，神经母细胞瘤x胶质瘤杂交细胞NG108-15在神经元分化过程中积累了脂褐素样自发荧光物质。脂褐素样自发荧光物质的最大发射波长在500至550 nm之间。酸性磷酸酶和过碘酸希夫染色阳性的颗粒增加，NG108-15细胞中的自发荧光颗粒也增加。巯基蛋白酶抑制剂N-(L-3-反式-羧基环氧乙烷-2-羰基)-L-亮氨酸-4-氨基丁酰胺（E-64）和乙酰-Leu-Leu-Arg（亮抑酶肽）显著加速了NG108-15细胞中脂褐素样自发荧光物质的积累。另一方面，溶酶体巯基蛋白酶组织蛋白酶B、C和L的活性在神经元分化过程中增加。细胞中的蛋白质含量随着神经元分化而逐渐增加，当E-64抑制蛋白水解时，这种增加显著加速。这些结果表明，脂褐素样自发荧光物质含有肽类物质作为成分，并表明神经元分化过程中巯基蛋白酶水解活性的增加不足以水解肽类底物，导致NG108-15细胞中自发荧光物质的积累。
