Oxidative Stress Impact on Barrier Function of Porcine Angular Aqueous Plexus Cell Monolayers
Oxidative Stress Impact on Barrier Function of Porcine Angular Aqueous Plexus Cell Monolayers
复制标题
氧化应激对猪角水丛细胞单层屏障功能的影响
DOI：
10.1167/iovs.12-11435
复制
发表时间：
2013-07-01
影响因子：
4.4
通讯作者：
Sun, Xinghuai
中科院分区：
文献类型：
--
作者：
Lei, Yuan;Stamer, William D.;Sun, Xinghuai
目的。我们的目标是研究慢性氧化应激对猪眼角房水丛（AAP，功能相当于人类施累姆氏管）内皮细胞的影响。方法。使用嘌呤霉素选择从猪流出组织中差异分离 AAP 细胞。猪 AAP 细胞的汇合培养物在生理 (5% O-2) 或高氧条件 (40% O-2) 下生长 2 周，以模拟与衰老相关的氧化应激升高。测量细胞生长速率、大小、跨内皮电阻（TEER）和水力传导率（HC）。监测衰老相关β-半乳糖苷酶和DNA损伤标记物8-羟基-20-脱氧鸟苷（8-OHdG）的表达，并通过免疫荧光染色和Western blot检测细胞骨架和细胞间粘附蛋白的水平，如F-肌动蛋白、磷酸肌球蛋白轻链（磷酸-MLC）、occludin、claudin-5、ZO-1、β-连环蛋白和VE-钙粘蛋白。印迹分析。结果。数据显示，慢性高氧从第3天开始抑制细胞生长速度，细胞大小增加18.2%+/-5.1%，细胞β-半乳糖苷酶和8-OHdG染色呈阳性。与对照组相比，高氧导致 TEER 显着增加 30%（P < 0.05，n = 6）。当以4 mm Hg从基底到顶端方向灌注时，对照组和高氧组AAP细胞的HC分别为1.97 +/- 0.12和1.54 +/- 0.13 μL/mm Hg/min/cm(2)（P < 0.05，n = 6）。通过免疫荧光和蛋白质印迹分析，与对照组相比，应激细胞表达显着更丰富的 F-肌动蛋白、磷酸-MLC、occludin、claudin-5、b-连环蛋白和 VE-钙粘蛋白。结论。 AAP 细胞长期暴露于氧化应激下，细胞单层通透性降低，细胞骨架和细胞间粘附蛋白表达上调；这表明，随着年龄的增长和氧化应激的增加，施累姆氏管水平的阻力也会增加。
PURPOSE. Our goal was to investigate the effect of chronic oxidative stress on angular aqueous plexus (AAP, functional equivalent to human Schlemm's canal) endothelial cells from porcine eyes.METHODS. AAP cells were differentially isolated from porcine outflow tissues using puromycin selection. Confluent cultures of porcine AAP cells were grown for 2 weeks in physiological (5% O-2) or hyperoxic conditions (40% O-2) to model elevated oxidative stress associated with ageing. Cell growth rate, size, transendothelial electrical resistance (TEER), and hydraulic conductivity (HC) were measured. The expression of senescence-associated beta-galactosidase and DNA damage marker 8-hydroxy-20-deoxyguanosine (8-OHdG) was monitored, and the levels of cytoskeletal and cell-cell adhesion proteins such as F-actin, phospho-myosin light chain (phosphor-MLC), occludin, claudin-5, ZO-1, beta-catenin, and VE-cadherin were measured by immunofluorescence staining and Western blot analysis.RESULTS. Data showed that chronic hyperoxia inhibited cell growth rate from day 3 onward, the cell size increased by 18.2% +/- 5.1%, and cells stained positive for beta-galactosidase and 8-OHdG. Hyperoxia resulted in a significant 30% increase in TEER compared with the control group (P < 0.05, n = 6). When perfused in the basal-to-apical direction at 4 mm Hg, HC of AAP cells was 1.97 +/- 0.12 and 1.54 +/- 0.13 mu L/mm Hg/min/cm(2) in control and hyperoxia groups, respectively (P < 0.05, n = 6). Stressed cells expressed a significantly greater abundance of F-actin, phospho-MLC, occludin, claudin-5, b-catenin, and VE-cadherin compared to the control group by both immunofluorescence and Western blot analyses.CONCLUSIONS. Chronic exposure of AAP cells to oxidative stress decreased cell monolayer permeability and up-regulated cytoskeletal and cell-cell adhesion protein expression; suggesting that, with age and increased oxidative stress, resistance at the level of Schlemm's canal increases.