缺氧对人视网膜色素上皮细胞蛋白质表达的影响

目的 为揭示缺氧对视网膜色素上皮(RPE)细胞的影响及与脉络膜新生血管(CNV)生成的关系提供实验依据.方法 实验研究.分别从对数生长期的正常及缺氧状态人RPE细胞中提取蛋白样本后行等电聚焦电泳(IEF),随后采用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)进行第二次分离.采用考马斯亮蓝染色法对凝胶进行显色固定,Image Master 2D Elite软件行分析配比,筛选出正常和缺氧RPE细胞表达明显差异的蛋白质斑点,随机选取差异斑点进行质谱分析,数据经蛋白质组数据库检索得出蒡异表达蛋白.结果 正常组分离蛋白斑点578个,组内匹配率92.90%;缺氧组559个,匹配率91.41%;两组间蛋白斑点匹配率为85.47%.发现volume值改变超过2倍的点共92个,其中32个点表达卜调,60个点表达下调.选择7个蛋白斑点行质谱分析,成功鉴定5种差异表达蛋白质(3个斑点为相同的蛋白):表达上调为热休克蛋白70、热休克蛋白60;表达下调的为β肌动蛋白、β微管蛋白以及过氧化还原蛋白3.结论 缺氧状态下RPE细胞表达差异蛋白可引起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、保持内部结构有序性的能力下降;本研究首次发现人RPE细胞在缺氧状态下热休克蛋白60表达上调,此蛋白的进一步研究可能能够发现新的CNV致病途径;蛋白质组学方法为CNV相关疾病的研究提供了-个很好的参考平台.

Screening and identification of abnormal expression proteins from hypoxia human retinal pigment epithelium cell in vitro

Objective To screen and identify the abnormal proteins expressed by hypoxia human retinal pigment epithelium (RPE) in vitro. Methods 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-offlight mass spectrometry (MALDI-TOF/MS). Results Five hunderd 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 ≥ 2.0 times. 7 spots were selected randomly for MS, and 5 proteins were identified successfully: HSP70 and HSP60 up-regulated; while β-actin, β-tubulin and proxiredoxin 3 down-regulated. Conclusions 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.