Status of mitochondria in living human fibroblasts during growth and senescence in vitro: use of the laser dye rhodamine 123.
Status of mitochondria in living human fibroblasts during growth and senescence in vitro: use of the laser dye rhodamine 123.
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DOI：
10.1083/jcb.91.2.392
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发表时间：
1981-11
期刊：
影响因子：
--
通讯作者：
Korczack LB
中科院分区：
文献类型：
--
作者：
Goldstein S;Korczack LB
罗丹明123是一种荧光激光染料，选择性地进入活细胞的线粒体，用于检查早期传代（年轻），晚期传代（老年）和早衰的人成纤维细胞的线粒体形态。在生长（对数中期）和汇合阶段，线粒体在所有细胞类型中很容易观察到。在汇合时的所有细胞株中，线粒体变得更短，更随机排列，并形成更高比例的珠状形式。用Tevenel（一种抑制线粒体蛋白质合成的氯霉素类似物）处理细胞6天，导致线粒体明显耗竭和弥漫的背景荧光。氰化物产生了一个快速释放预先加载的线粒体荧光，然后分离和杀死细胞。秋水仙胺引起线粒体的随机卷曲和破碎，特别是在融合期。在对数中期和汇合状态或这些处理后，三种细胞株的线粒体中没有明显差异。与对数中期阶段相比，在汇合的所有细胞株中，装载罗丹明123后的丁醇可提取荧光较低。在汇合时，所有三种细胞株在零时和洗脱后24 h内的罗丹明含量相似。在对数中期，年轻的细胞最初含有更多的罗丹明，并以该顺序比老年或早衰细胞更快地失去它。数据表明，老年和早衰成纤维细胞中线粒体的形态或数量没有明显的紊乱，但在对数中期，这些细胞中质子动力明显降低，这可能是生长限制。
Rhodamine 123, a fluorescent laser dye that is selectively taken up into mitochondria of living cells, was used to examine mitochondrial morphology in early-passage (young), late-passage (old), and progeric human fibroblasts. Mitochondria were readily visualized in all cell types during growth (mid-log) and confluent stages. In all cell strains at confluence, mitochondria became shorter, more randomly aligned, and developed a higher proportion of bead-like forms. Treatment of cells for six days with Tevenel, a chloramphenicol analog that inhibits mitochondrial protein synthesis, brought about a marked depletion of mitochondria and a diffuse background fluorescence. Cyanide produced a rapid release of preloaded mitochondrial fluorescence followed by detachment and killing of cells. Colcemid caused a random coiling and fragmentation of mitochondria particularly in the confluent stage. No gross differences were discernible in mitochondria of the three cell strains in mid-log and confluent states or after these treatments. Butanol-extractable fluorescence after loading with rhodamine 123 was lower in all cell strains in confluent compared to mid-log stages. At confluence all three cell strains had similar rhodamine contents at zero-time and after washout up to 24 h. At the mid-log stage, young cells contained more rhodamine initially and lost it more rapidly than old or progeria cells, in that order. The data indicate no gross derangement in the morphology or number of mitochondria in old and progeria fibroblasts but there is a reduction of protonmotive force evident in these cells at the mid-log stage that may be growth limiting.