Increased stress-induced generation of reactive oxygen species and apoptosis in human keratoconus fibroblasts
Increased stress-induced generation of reactive oxygen species and apoptosis in human keratoconus fibroblasts
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DOI：
10.1167/iovs.05-0828
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发表时间：
2006-05-01
影响因子：
4.4
通讯作者：
Kenney, M. Cristina
中科院分区：
文献类型：
--
作者：
Chwa, Marilyn;Atilano, Shari R.;Kenney, M. Cristina
目的。研究圆锥角膜（KC）成纤维细胞培养是否增加活性氧（ROS）的产生，是否更容易受到应激相关的挑战。正常（n = 9）和KC （n = 10）间质成纤维细胞培养在中性或低ph条件下，有或没有过氧化氢。用荧光底物法测定过氧化氢酶活性。超氧化物和活性氧/活性氮（RNS）的产生分别用胺反应绿色染料法和2',7'-二氯二氢荧光素二乙酸（H(2)DCFDA）染料法测定。用染料排除法分析细胞活力。用Caspase荧光抑制剂（FLICA）测定Caspase 3活性。采用阳离子（绿色）染料测定线粒体膜电位（δ Psi m）。高浓度H2O2使KC成纤维细胞的超氧化物和ROS/RNS产量（分别为6.2倍，P < 0.001和1.8倍，P < 0.001）和过氧化氢酶活性（P < 0.01）高于正常培养（P = 0.16）。低ph胁迫后，KC成纤维细胞与正常成纤维细胞相比，ROS/RNS水平保持较高（3.3倍，P < 0.02）， caspase-3活性升高（7.5倍，P < 0.02）， δ Psi - m降低（2.6倍，P < 0.04），细胞活力降低（37%,P < 0.005 vs. 20%, P < 0.27）。在相同的条件下，KC成纤维细胞增加了ROS/RNS的基础生成，并且比正常成纤维细胞更容易受到应激挑战（低ph和/或H2O2条件）。此外，应激的KC成纤维细胞具有与完整的KC角膜相似的特征（过氧化氢酶活性、ROS产生和凋亡增加）。这些特性可能在KC的发病机制中起作用。
PURPOSE. To determine whether keratoconus ( KC) corneal fibroblast cultures have increased reactive oxygen species ( ROS) production and are more susceptible to stress-related challenges.METHODS. Normal (n = 9) and KC ( n = 10) stromal fibroblast cultures were incubated in either neutral- or low-pH conditions, with or without hydrogen peroxide. Catalase activities were measured with a fluorescent substrate assay. Superoxide and ROS/reactive nitrogen species (RNS) productions were determined with an amine-reactive green-dye assay and 2', 7'-dichlorodihydrofluorescein diacetate (H(2)DCFDA) dye assay, respectively. Cell viability was analyzed by a dye-exclusion assay. Caspase 3 activity was measured by a fluorochrome inhibitor of caspase (FLICA) assay. A cationic ( green) dye was used to measure the mitochondrial membrane potential (Delta Psi m).RESULTS. KC fibroblasts had increased superoxide and ROS/RNS production (6.2-fold, P < 0.001 and 1.8-fold, P < 0.001, respectively) and catalase activity ( P < 0.01) with higher concentrations of H2O2 compared with normal cultures ( P = 0.16). After a low-pH stress challenge, KC fibroblasts maintained higher ROS/RNS levels (3.3-fold, P < 0.02), showed higher caspase-3 activity (7.5-fold, P < 0.02) and decreased Delta Psi m ( 2.6-fold, P < 0.04), and had decreased cell viability (37%, P < 0.005 vs. 20%, P < 0.27) compared with normal fibroblasts.CONCLUSIONS. Under identical conditions, KC fibroblasts had increased basal generation of ROS/RNS and were more susceptible to stressful challenges (low-pH and/or H2O2 conditions) than were normal fibroblasts. In addition, the stressed KC fibroblasts possessed characteristics similar to those found in the intact KC corneas ( increased catalase activity, ROS production, and apoptosis). These properties may play a role in the pathogenesis of KC.