He Shou-zhi, Hou Bao-ke, Tang Wei-qiang, Ding Zhen-qiang
Department of Ophthalmology, General Hospital of PIA, Beijing 100853, China.
Zhonghua Yan Ke Za Zhi. 2008 Jun;44(6):534-9.
To screen and identify the abnormal proteins expressed by hypoxia human retinal pigment epithelium (RPE) in vitro.
It was a experimental study. Protein of normal/hypoxia human RPE cells in exponential phase of growth was extracted, and frozen by liquid nitrogen for proteome study. All samples were performed isoelectric focusing electrophoresis (IEF), separated by isoelectric point (pI); progressed sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) and separated by protein molecular weight (Mr). 2D-gels revealed by coomassie brilliant blue, analyzed by Image Master 2D Elite. Differential proteins spots were screened in normal/hypoxia RPE cell. For identification of differential proteins, peptide masses were analyzed by matrix-assisted laser desorption ionization time-of flight mass spectrometry (MALDI-TOF/MS).
Five hundred and seventy-eight protein spots were obtained in normal group (matching rate within group was 92.90%), and hypoxia group 559 (91.41%). The average matching rate between two groups was 85.47%. There were 32 differential protein spots, which volume value changed > or = 2.0 times. 7 spots were selected randomly for MS, and 5 proteins were identified successfully: HSP70 and HSP60 up-regulated; while beta-actin, beta-tubulin and peroxiredoxin 3 down-regulated.
Findings of the in vitro study provide the evidence for expression changes of many proteins in RPE cell with hypoxia state; some of those regulated proteins can result in increasing stress capability obviously in cells, while the major cytoskeletal proteins down-regulated, and the ability of sustained-shapes, keeping order internal structure in cells decreasing. Proteome analysis provides a useful platform in systematic screening of various protein expressions in CNV related diseases.
筛选并鉴定体外培养的缺氧人视网膜色素上皮（RPE）细胞中表达异常的蛋白质。
本研究为实验性研究。提取处于生长指数期的正常/缺氧人RPE细胞的蛋白质，液氮冻存用于蛋白质组学研究。所有样本均进行等电聚焦电泳（IEF），按等电点（pI）分离；再进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE），按蛋白质分子量（Mr）分离。考马斯亮蓝染色显示二维凝胶，用Image Master 2D Elite软件分析。筛选正常/缺氧RPE细胞中的差异蛋白质斑点。为鉴定差异蛋白质，采用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF/MS）分析肽质量。
正常组获得578个蛋白质斑点（组内匹配率为92.90%），缺氧组获得559个（91.41%）。两组间平均匹配率为85.47%。有32个差异蛋白质斑点，其体积值变化≥2.0倍。随机选取7个斑点进行质谱分析，成功鉴定出5种蛋白质：热休克蛋白70（HSP70）和热休克蛋白60（HSP60）上调；而β-肌动蛋白、β-微管蛋白和过氧化物酶体增殖物激活受体3（peroxiredoxin 3）下调。
体外研究结果为缺氧状态下RPE细胞中多种蛋白质表达变化提供了证据；其中一些受调控的蛋白质可使细胞应激能力明显增强，而主要细胞骨架蛋白下调，细胞维持形态、保持内部结构有序的能力下降。蛋白质组分析为系统性筛选CNV相关疾病中各种蛋白质表达提供了有用平台。
