Chen Lin, Dentchev Tzvete, Wong Robert, Hahn Paul, Wen Rong, Bennett Jean, Dunaief Joshua L
F. M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA, USA.
Mol Vis. 2003 Apr 30;9:151-8.
Oxidative stress plays a role in the photic injury model of retinal degeneration and in age-related macular degeneration. Our preliminary microarray analysis of retinal gene expression upon photic injury suggested increased expression of ceruloplasmin, a ferroxidase that could reduce retinal oxidative stress. Patients with acerul oplasminemia have retinal degeneration, indicating that ceruloplasmin is necessary for maintenance of retinal health. The purpose of this study was to determine whether retinal ceruloplasmin is upregulated following photo-oxidation, to localize ceruloplasmin protein, and to determine which ceruloplasmin isoform is present in the retina.
Balb/c mice were exposed to bright white light for seven hours. TUNEL labeling was used to detect photoreceptor apoptosis. At several intervals after the light injury, retinal ceruloplasmin was studied by quantitative PCR, immunohistochemistry, and western analysis. Expression of the secreted and expression of the membrane-anchored glycosyl phosphatidyl inositol (GPI) linked forms of ceruloplasmin were assesed in rat retina using primers specific for each form. Vitreous ceruloplasmin was detected by immunohistochemistry in Balb/c mouse eyes and by western analysis of aspirated vitreous from post-mortem human eyes.
Retinal ceruloplasmin mRNA was upregulated eight-fold following photic injury. Ceruloplasmin protein was detected throughout normal retinas by immunohistochemistry, with a specific increase in Muller cell labeling following photic injury. Western analysis confirmed an increase in ceruloplasmin protein following photic injury and revealed eight-fold more ceruloplasmin protein in normal retina than in brain. The mRNAs for both the secreted and GPI linked forms of ceruloplasmin were detected by RT-PCR in the retina. Ceruloplasmin protein was detected by western analysis of normal human vitreous and was increased in mouse vitreous following photic injury.
Ceruloplasmin, a retinal ferroxidase, is upregulated at the mRNA and protein levels upon light damage. The increased protein is primarily in Muller cells. Ceruloplasmin is considerably more abundant in retina than in brain. The retina expresses both the GPI-linked and secreted forms of ceruloplasmin, and since vitreous ceruloplasmin increases following photic injury, some of the retinal ceruloplasmin may be secreted into the vitreous. Ceruloplasmin may protect the retina from oxidative stress by decreasing the amount of ferrous iron available to produce reactive oxygen species.
氧化应激在视网膜变性的光损伤模型以及年龄相关性黄斑变性中起作用。我们对光损伤后视网膜基因表达进行的初步微阵列分析表明，铜蓝蛋白（一种可降低视网膜氧化应激的铁氧化酶）的表达增加。无铜蓝蛋白血症患者会出现视网膜变性，这表明铜蓝蛋白对于维持视网膜健康是必需的。本研究的目的是确定光氧化后视网膜铜蓝蛋白是否上调，定位铜蓝蛋白蛋白，并确定视网膜中存在哪种铜蓝蛋白异构体。
将Balb/c小鼠暴露于强光下7小时。TUNEL标记用于检测光感受器凋亡。在光损伤后的几个时间点，通过定量PCR、免疫组织化学和western分析研究视网膜铜蓝蛋白。使用针对每种形式的特异性引物，在大鼠视网膜中评估铜蓝蛋白的分泌形式和膜锚定糖基磷脂酰肌醇（GPI）连接形式的表达。通过免疫组织化学在Balb/c小鼠眼中检测玻璃体液中的铜蓝蛋白，并通过对死后人类眼睛吸出的玻璃体液进行western分析来检测。
光损伤后视网膜铜蓝蛋白mRNA上调了8倍。通过免疫组织化学在整个正常视网膜中检测到铜蓝蛋白蛋白，光损伤后Muller细胞标记有特异性增加。western分析证实光损伤后铜蓝蛋白蛋白增加，并显示正常视网膜中的铜蓝蛋白蛋白比脑中多8倍。通过RT-PCR在视网膜中检测到铜蓝蛋白的分泌形式和GPI连接形式的mRNA。通过对正常人玻璃体液进行western分析检测到铜蓝蛋白蛋白，光损伤后小鼠玻璃体液中的铜蓝蛋白蛋白增加。
铜蓝蛋白，一种视网膜铁氧化酶，在光损伤后在mRNA和蛋白水平上上调。增加的蛋白主要存在于Muller细胞中。视网膜中的铜蓝蛋白比脑中丰富得多。视网膜表达GPI连接形式和分泌形式的铜蓝蛋白，并且由于光损伤后玻璃体液中的铜蓝蛋白增加，一些视网膜铜蓝蛋白可能分泌到玻璃体液中。铜蓝蛋白可能通过减少可用于产生活性氧的亚铁离子量来保护视网膜免受氧化应激。
