Chwa Marilyn, Atilano Shari R, Hertzog Dieter, Zheng Hong, Langberg Jonathan, Kim Dae W, Kenney M Cristina
Department of Ophthalmology, University of California, Irvine Medical Center, Orange, California 92868, USA.
Invest Ophthalmol Vis Sci. 2008 Oct;49(10):4361-9. doi: 10.1167/iovs.08-1969. Epub 2008 May 30.
It is unclear whether the oxidative damage found in keratoconus (KC) corneas results from innate defects of corneal fibroblasts or is due to excessive environmental challenges encountered by the patient with KC. The purpose of this study was to explore whether KC cells have inherent, exaggerated hypersensitivity to oxidative stressors.
Normal and KC corneal stromal fibroblasts were incubated in neutral or low-pH conditions, with or without hydrogen peroxide (H(2)O(2)). Reactive oxygen/nitrogen species (ROS/RNS) production was measured with 2',7'-dichlorodihydrofluorescein diacetate dye. Caspase-9 and -12 activities were measured by fluorochrome inhibitor to caspase (FLICA) assays. Long-extension polymerase chain reaction (LX-PCR) was used to amplify mtDNA. RNA was extracted, full-length cDNA synthesized, and PCR performed for mitochondria-encoded genes. Mitochondrial membrane potential (DeltaPsim) was measured by a cationic dye assay.
In neutral pH conditions, KC fibroblasts had increased ROS production (P = 0.047), higher RNA levels for cytochrome c oxidase (complex IV) subunit II (P < 0.05), and decreased cathepsin K RNA (P = 0.04) compared with levels in normal cultures. In low-pH conditions, KC fibroblasts had decreased DeltaPsim (P = 0.015) and increased activation of caspase-9 (P = 0.013) and -12 (P = 0.01) compared with normal cultures. Changes in DeltaPsim were independent of cathepsin inhibition. The combination of low-pH+H(2)O(2) treatment degraded intact mtDNA and decreased the mtDNA-to-nuclear DNA ratio.
Cultured KC fibroblasts have an inherent, hypersensitive response to oxidative stressors that involves mitochondrial dysfunction and mtDNA damage. KC fibroblast hypersensitivity may play a role in the development and progression of keratoconus.
圆锥角膜（KC）角膜中发现的氧化损伤是源于角膜成纤维细胞的先天性缺陷，还是由于KC患者面临的过度环境挑战，目前尚不清楚。本研究的目的是探讨KC细胞是否对氧化应激源具有内在的、过度的超敏反应。
将正常和KC角膜基质成纤维细胞在中性或低pH条件下培养，添加或不添加过氧化氢（H₂O₂）。用2',7'-二氯二氢荧光素二乙酸酯染料测量活性氧/氮物质（ROS/RNS）的产生。通过荧光素酶抑制caspase（FLICA）测定法测量caspase-9和-12的活性。使用长延伸聚合酶链反应（LX-PCR）扩增线粒体DNA（mtDNA）。提取RNA，合成全长cDNA，并对线粒体编码基因进行PCR。通过阳离子染料测定法测量线粒体膜电位（ΔΨm）。
在中性pH条件下，与正常培养物相比，KC成纤维细胞的ROS产生增加（P = 0.047），细胞色素c氧化酶（复合体IV）亚基II的RNA水平更高（P < 0.05），组织蛋白酶K RNA水平降低（P = 0.04）。在低pH条件下，与正常培养物相比，KC成纤维细胞的ΔΨm降低（P = 0.015），caspase-9（P = 0.013）和-12（P = 0.01）的活化增加。ΔΨm的变化与组织蛋白酶抑制无关。低pH + H₂O₂处理的组合降解了完整的mtDNA并降低了mtDNA与核DNA的比率。
培养的KC成纤维细胞对氧化应激源具有内在的超敏反应，涉及线粒体功能障碍和mtDNA损伤。KC成纤维细胞超敏反应可能在圆锥角膜的发生和发展中起作用。
