UV dose-dependent caspase activation in a corneal epithelial cell line
UV dose-dependent caspase activation in a corneal epithelial cell line
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DOI：
10.1076/ceyr.28.2.85.26237
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发表时间：
2004-02-01
影响因子：
2
通讯作者：
Tsubota, K
中科院分区：
文献类型：
--
作者：
Shimmura, S;Tadano, K;Tsubota, K
目的。表征 SV 40 永生化角膜上皮细胞中半胱天冬酶激活和细胞死亡的 UVB 辐射依赖性模式。方法。永生化人角膜上皮细胞死亡。细胞（T-HCEC）通过暴露于低（50 mJ/cm(2)）和高（450 mJ/cm(2)）剂量的UVB来诱导。使用细胞死亡标记物碘化丙啶（PI）通过荧光显微镜检查细胞死亡形态。通过 DNA 片段化测定分析 T-HCEC 的凋亡，并通过荧光光度法测量 caspase 3 和 9 的酶活性。使用荧光标记物罗丹明 123 通过流式细胞术测量线粒体内膜电位的变化。在辐射后，测量 T-HCEC 胞质部分中细胞色素 c（半胱天冬酶 9 的上游触发器）的重新分布。结果。 PI染色显示细胞核的碎片染色模式与低剂量UVB照射的分离细胞的凋亡一致。接受高剂量 UVB 的细胞表现出圆形、边界清晰的细胞核染色。流式细胞术显示高剂量组中出现不可逆的线粒体损伤，表现为罗丹明 123 荧光水平降低。低剂量组细胞线粒体内膜电位完整，胞质细胞色素c增加，DNA断裂率和caspase激活率均显着高于高剂量组。结论。低剂量UVB引起细胞色素c重新分布、半胱天冬酶激活和角膜上皮细胞凋亡，而在高剂量UVB照射下则没有观察到这种情况。
Purpose. To characterize the UVB radiation-dependent patterns of caspase activation and cell death in SV 40 immortalized corneal epithelial cells.Methods. Cell death in immortalized human corneal epithelial. cells (T-HCEC) was induced by exposure to low (50 mJ/cm(2)) and high (450 mJ/cm(2)) 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.Results. 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.Conclusion. 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.