Jou Mei-Jie, Jou Shuo-Bin, Chen Hung-Ming, Lin Chi-Hung, Peng Tsung-I
Department of Physiology and Pharmacology, Chang Gung University, Tao-Yuan, Taiwan, ROC.
J Biomed Sci. 2002;9(6 Pt 1):507-16. doi: 10.1159/000064723.
Laser irradiation-induced phototoxicity has been intensively applied in clinical photodynamic therapy for the treatment of a variety of tumors. However, the precise laser damage sites as well as the underlying mechanisms at the subcellular level are unknown. Using a mitochondrial fluorescent marker, MitoTracker Green, severe mitochondrial swelling was noted in laser-irradiated rat brain astrocytes. Nucleus condensation and fragmentation revealed by propidium iodide nucleic acid staining indicated that laser-irradiated cells died from apoptosis. Using an intracellular reactive oxygen species (ROS) fluorescent dye, 2',7'-dichlorofluorescin diacetate, heterogeneous distribution of ROS inside astrocytes was observed after laser irradiation. The level of ROS in the mitochondrial compartment was found to be higher than in other parts of the cell. With another ROS fluorescent dye, dihydrorhodamine-123, and time-lapse laser scanning confocal microscopy, a substantial increase in mitochondrial ROS (mROS) was visualized in visible laser-irradiated astrocytes. The antioxidants melatonin and vitamin E largely attenuated laser irradiation-induced mROS formation and prevented apoptosis. Cyclosporin A (CsA), a mitochondrial permeability transition (MPT) blocker, did not prevent visible laser irradiation-induced mROS formation and apoptosis. In conclusion, mROS formation contributes significantly to visible laser irradiation-induced apoptosis via an MPT-independent pathway.
激光照射诱导的光毒性已被广泛应用于临床光动力疗法以治疗多种肿瘤。然而，亚细胞水平上精确的激光损伤位点以及潜在机制尚不清楚。使用线粒体荧光标记物MitoTracker Green，在激光照射的大鼠脑星形胶质细胞中观察到严重的线粒体肿胀。碘化丙啶核酸染色显示的细胞核浓缩和碎片化表明激光照射的细胞死于凋亡。使用细胞内活性氧（ROS）荧光染料2',7'-二氯荧光素二乙酸酯，激光照射后观察到星形胶质细胞内ROS的异质性分布。发现线粒体区室中的ROS水平高于细胞的其他部分。使用另一种ROS荧光染料二氢罗丹明-123以及延时激光扫描共聚焦显微镜，在可见激光照射的星形胶质细胞中观察到线粒体ROS（mROS）大量增加。抗氧化剂褪黑素和维生素E在很大程度上减弱了激光照射诱导的mROS形成并防止了凋亡。线粒体通透性转换（MPT）阻断剂环孢素A（CsA）不能防止可见激光照射诱导的mROS形成和凋亡。总之，mROS的形成通过不依赖MPT的途径对可见激光照射诱导的凋亡有显著贡献。
