Gerland Luc-Marie, Peyrol Simone, Lallemand Christophe, Branche Robert, Magaud Jean-Pierre, Ffrench Martine
INSERM U 453, Biologie des Gènes Suppresseurs de Tumeur, Faculté de Médecine, 8 avenue Rockefeller et Centre Léon Bérard, 69373 Lyon 08, France.
Exp Gerontol. 2003 Aug;38(8):887-95. doi: 10.1016/s0531-5565(03)00132-3.
Replicative senescence appears after a finite number of cell divisions. After proliferation has ceased, senescent cells remain viable for long periods and metabolic modifications are observed such as lipofuscin accumulation. In order to understand this phenomenon, we examined the emergence of subcellular modifications corresponding to autophagy in MRC5 normal human fibroblasts. An increase of monodansylcadaverine fluorescence, a specific marker of autophagy, in aging compared to young fibroblasts was observed (p<0.0001). The increase of autophagic vacuoles in aging fibroblasts was confirmed by electron microscopy. We compared young versus senescent fibroblasts and showed that autophagic vacuoles, already present in young cells, became larger in senescent fibroblasts with a significant relative increase of inclusion area with respect to measured cell area (p=0.0041). However, autophagy-associated-gene expression remained stable in senescent compared to young fibroblasts, suggesting that the autophagy process per se is not enhanced. In parallel, transmission electron microscopy analysis showed that beta-galactosidase activity distribution was modified by aging: beta-galactosidase (an enzyme linked to lysosome) was scattered in young fibroblasts, but clustered at the level of autophagic vacuoles in senescent fibroblasts, suggesting a predominance of autolysosomes at this stage. These results support the hypothesis that, during fibroblast aging, the increase of autophagic vacuoles, as well as that of beta-galactosidase activity, may be associated to an increase of lysosomal mass and to an accumulation of degradative autolysosomes with lipofuscin. This phenomenon could be involved in the death of senescent fibroblasts.
复制性衰老出现在有限次数的细胞分裂之后。增殖停止后，衰老细胞可长期存活，并观察到代谢改变，如脂褐素积累。为了理解这一现象，我们研究了MRC5正常人成纤维细胞中与自噬相对应的亚细胞修饰的出现情况。与年轻成纤维细胞相比，衰老细胞中自噬的特异性标志物单丹磺酰尸胺荧光增加（p<0.0001）。衰老成纤维细胞中自噬泡的增加通过电子显微镜得以证实。我们比较了年轻与衰老的成纤维细胞，结果显示，自噬泡在年轻细胞中就已存在，在衰老成纤维细胞中变得更大，其包含物面积相对于所测细胞面积有显著相对增加（p=0.0041）。然而，与年轻成纤维细胞相比，衰老细胞中自噬相关基因的表达保持稳定，这表明自噬过程本身并未增强。同时，透射电子显微镜分析表明，衰老会改变β-半乳糖苷酶的活性分布：β-半乳糖苷酶（一种与溶酶体相关的酶）在年轻成纤维细胞中呈散在分布，但在衰老成纤维细胞的自噬泡水平聚集，这表明在此阶段自溶酶体占主导。这些结果支持了这样一种假说，即在成纤维细胞衰老过程中，自噬泡的增加以及β-半乳糖苷酶活性的增加可能与溶酶体质量的增加以及含有脂褐素的降解性自溶酶体的积累有关。这种现象可能参与了衰老成纤维细胞的死亡。
