Shimmura Shigeto, Tadano Kimie, Tsubota Kazuo
Department of Ophthalmology, Tokyo Dental College, Chiba, Japan.
Curr Eye Res. 2004 Feb;28(2):85-92. doi: 10.1076/ceyr.28.2.85.26237.
To characterize the UVB radiation-dependent patterns of caspase activation and cell death in SV 40 immortalized corneal epithelial cells.
Cell death in immortalized human corneal epithelial cells (T-HCEC) was induced by exposure to low (50 mJ/cm2) and high (450 mJ/cm2) doses of UVB. Cell death morphology was examined by fluorescence microscopy using the cell death marker propidium iodide (PI). Apoptosis of T-HCEC was analyzed by DNA fragmentation assays, and enzyme activity was measured for caspase 3 and 9 by fluorophotometry. Changes in mitochondrial inner membrane potential were measured by flow cytometry using the fluorescent marker, rhodamine 123. Redistribution of cytochrome c, the upstream trigger of caspase 9, was measured in the cytosol fraction of T-HCEC following irradiation.
PI staining revealed a fragmented staining pattern of the nucleus consistent with apoptosis in detached cells irradiated with low-dose UVB, while cells receiving high dose UVB demonstrated round, well bordered staining of the nucleus. Flow cytometry revealed irreversible mitochondrial damage in the high dose group shown by decreased levels of rhodamine 123 fluorescence. Cells in the low-dose group had intact mitochondrial inner membrane potential, increased cytosolic cytochrome c, and showed a significantly higher rate of DNA fragmentation and caspase activation than the high dose group.
Low dose UVB caused cytochrome c redistribution, caspase activation and apoptosis of corneal epithelial cells, which was not observed at high irradiation levels of UVB.
描述SV40永生化角膜上皮细胞中紫外线B（UVB）辐射依赖性半胱天冬酶激活和细胞死亡模式。
通过暴露于低剂量（50 mJ/cm2）和高剂量（450 mJ/cm2）的UVB诱导永生化人角膜上皮细胞（T-HCEC）发生细胞死亡。使用细胞死亡标记碘化丙啶（PI）通过荧光显微镜检查细胞死亡形态。通过DNA片段化分析检测T-HCEC的凋亡，并通过荧光光度法测量半胱天冬酶3和9的酶活性。使用荧光标记罗丹明123通过流式细胞术测量线粒体内膜电位的变化。在照射后的T-HCEC细胞质部分中测量半胱天冬酶9的上游触发因子细胞色素c的重新分布。
PI染色显示，低剂量UVB照射的脱离细胞中，细胞核呈现与凋亡一致的碎片化染色模式，而接受高剂量UVB的细胞则显示细胞核呈圆形、边界清晰的染色。流式细胞术显示高剂量组中罗丹明123荧光水平降低，表明线粒体受到不可逆损伤。低剂量组细胞的线粒体内膜电位完整，细胞质细胞色素c增加，并且与高剂量组相比，DNA片段化和半胱天冬酶激活率显著更高。
低剂量UVB导致角膜上皮细胞的细胞色素c重新分布、半胱天冬酶激活和凋亡，而在高剂量UVB照射水平下未观察到这些现象。
