Department of Ophthalmology, University of California, Irvine, California, USA.
Invest Ophthalmol Vis Sci. 2010 Feb;51(2):1164-70. doi: 10.1167/iovs.09-3443. Epub 2009 Oct 15.
To assess oxysterol-induced mitochondrial DNA (mtDNA) damage and mitochondrial dysfunction in cultured human retinal pigment epithelial cells (ARPE- 19).
ARPE-19 cultures were exposed for 6 and 24 hours to 40 microg/mL 7-ketocholesterol (7kCh), and total DNA was extracted. Long-extension polymerase chain reaction was performed to amplify the full-length mtDNA genome. The products were separated by electrophoresis on a 0.8% agarose gel stained with ethidium bromide. Superoxide and reactive oxygen/nitrogen species (ROS/RNS; hydrogen peroxide, peroxynitrite anions, and peroxyl radicals) were measured with an amine-reactive green-dye assay and 2',7'-dicholorodihydrofluorescein diacetate (H(2)DCFDA) dye assay, respectively. The changes in mitochondrial membrane potential (DeltaPsim) were measured with a cationic (green) dye assay. Western blot analysis was used to assess porins, a structural protein of the mitochondrial membranes.
The 7kCh-treated cultures showed significant increase in ROS/RNS production (P < 0.001) compared with untreated controls, but the superoxide levels were unchanged. The 7kCh-treated ARPE-19 cultures had diminished levels of the full-length 16.2-kb mtDNA band, a 2.2-fold decrease of the DeltaPsim compared with control cultures (P < 0.001), and decreased levels of porins.
7kCh causes significant damage to the full-length intact mtDNA and mitochondrial dysfunction in ARPE-19 cells. These observations suggest that the mitochondria and its DNA may be targets for oxysterol-induced oxidative stress and may play a role in the pathogenesis of retinal diseases.
评估氧化固醇诱导的人视网膜色素上皮细胞（ARPE-19）中线粒体 DNA（mtDNA）损伤和线粒体功能障碍。
将 ARPE-19 培养物暴露于 40μg/ml 7-酮胆固醇（7kCh）中 6 和 24 小时，并提取总 DNA。通过长延伸聚合酶链反应扩增全长 mtDNA 基因组。将产物在含有溴化乙锭的 0.8%琼脂糖凝胶上电泳分离。通过胺反应性绿色染料测定法和 2'，7'-二氯二氢荧光素二乙酸酯（H2DCFDA）染料测定法分别测量超氧化物和活性氧/氮物质（ROS/RNS；过氧化氢、过氧亚硝酸盐阴离子和过氧自由基）。通过阳离子（绿色）染料测定法测量线粒体膜电位（DeltaPsim）的变化。使用 Western blot 分析评估孔蛋白，这是线粒体膜的结构蛋白。
与未处理的对照组相比，7kCh 处理的培养物显示出明显增加的 ROS/RNS 产生（P < 0.001），但超氧化物水平不变。7kCh 处理的 ARPE-19 培养物的全长 16.2kb mtDNA 带水平显著降低，与对照培养物相比，DeltaPsim 降低了 2.2 倍（P < 0.001），并且孔蛋白水平降低。
7kCh 导致 ARPE-19 细胞中线粒体全长完整 mtDNA 明显受损和线粒体功能障碍。这些观察结果表明，线粒体及其 DNA 可能是氧化固醇诱导的氧化应激的靶标，并可能在视网膜疾病的发病机制中发挥作用。
