Cytoskeletal and morphologic impact of cellular oxidant injury.
Cytoskeletal and morphologic impact of cellular oxidant injury.
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发表时间：
1986-06
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通讯作者：
D. B. Hinshaw;Larry A. Sklar;Ben Bohl;Ingrid U. SCHRAUFSTATrER;P A Hyslop;Michael W. Rossi;R. Spragg;C G Cochrane
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作者：
D. B. Hinshaw;Larry A. Sklar;Ben Bohl;Ingrid U. SCHRAUFSTATrER;P A Hyslop;Michael W. Rossi;R. Spragg;C G Cochrane
在P388 D1细胞系中检查了氧化损伤中细胞形态学变化与细胞骨架之间的关系。用NBD-鬼笔毒肽（一种特异于丝状（F）肌动蛋白的荧光探针）染色的细胞的流式细胞术显示，在H2 O2损伤的细胞中，F肌动蛋白含量在3-4小时内显著增加。低至500 μ M的H2 O2剂量产生F肌动蛋白含量的持续增加。使用过氧化氢酶中断长时间H2 O2暴露的实验表明，15-30分钟是暴露于5 mM H2 O2的关键时期，这是F肌动蛋白持续增加以及膜起泡大幅增加和随后细胞死亡所必需的。F肌动蛋白与H2 O2损伤的增加，证实了使用电泳在丙烯酰胺凝胶的1%Triton X-100细胞骨架提取物从P388 D1细胞。扫描电子显微镜显示，除了形成大泡外，表面卷曲的主要损失。使用罗丹明鬼笔环肽的粘附细胞的荧光显微镜显示相当大的细胞变圆和重排的细胞F肌动蛋白暴露于H2 O2 30分钟。透射电子显微镜显示侧边聚集的F肌动蛋白束（微丝）在这段时间内发展。损伤2小时后可见线粒体和其他亚细胞器明显肿胀。在受损细胞中，与基质的表观附着面积显著减少。H2 O2损伤产生了显着增加F肌动蛋白与相关的微丝重排和细胞死亡前在P388 D1细胞系中的质膜的同时变化。
The relationship between changes in cell morphology and the cytoskeleton in oxidant injury was examined in the P388D1 cell line. Flow cytometry of cells stained with NBD-phallacidin, a fluorescent probe specific for filamentous (F) actin, revealed a substantial increase in F actin content in H2O2-injured cells over 3-4 hours. Doses of H2O2 as low as 500 microM produced sustained increases in F actin content. Experiments where catalase was used to interrupt H2O2 exposure over a long time course revealed 15-30 minutes to be the critical period of exposure to 5 mM H2O2 necessary for a sustained increase in F actin as well as large increases in membrane blebbing and later cell death. The increase in F actin with H2O2 injury was confirmed with the use of electrophoresis in acrylamide gels of 1% Triton X-100 cytoskeletal extracts from P388D1 cells. Scanning electron microscopy revealed major loss of surface convolutions in addition to the formation of blebs. Fluorescence microscopy of adherent cells using rhodamine phalloidin showed considerable cell rounding and rearrangement of cellular F actin by 30 minutes of exposure to H2O2. Transmission electron microscopy revealed side to side aggregation of F actin bundles (microfilaments) developing during this time. Considerable swelling of mitochondria and other subcellular organelles was seen after 2 hours of injury. The apparent area of attachment to the substrate was markedly diminished in injured cells. H2O2 injury produced a marked increase in F actin with an associated rearrangement of the microfilaments and simultaneous changes in the plasma membrane prior to cell death in the P388D1 cell line.