南方医科大学学报

南方医科大学学报 ›› 2016, Vol. 36 ›› Issue (05): 595-.

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Proteomic analysis of retinal proteins in rabbits following intravitreal PBS injection: analysis with tandem mass tag labeling coupled with LC-MS/MS

WANG Jiaming, LEI Bo   

  1. Department of Ophthalmology, the First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology, Chongqing Eye Institute, Chongqing 400016, China
  • 出版日期:2016-05-20 发布日期:2016-05-20

兔玻璃体腔注射PBS后视网膜差异蛋白的表达

汪家名，雷博   

  1. Department of Ophthalmology, the First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology, Chongqing Eye Institute, Chongqing 400016, China
  • Online:2016-05-20 Published:2016-05-20

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摘要： 目的研究家兔玻璃体腔内注射PBS后的视网膜功能变化及差异蛋白的表达情况以探索玻璃体腔注射的安全性。方法 将20只青紫蓝兔随机分为4组，A组不作任何处理，B至D组在兔右眼玻璃体腔内注射50 μL PBS。分别于注射后第0、4、7、14 天进行眼底照相和视网膜电图（ERG）检查，之后摘取眼球并分离视网膜。利用串联质谱标签（TMT）联合液相色谱-串联质谱联 用（LC-MS/MS）法检测注射组相对于空白对照组的视网膜蛋白变化情况。通过KOBAS及基于KEGG数据库分析明显变化的 蛋白富集通路，利用STRING得出明显变化蛋白间相互作用的功能网络图。结果注射组眼底照相结果与空白组相比无明显变 化，ERG结果显示视网膜功未受到影响。TMT蛋白质谱共检测出了6042种兔视网膜蛋白，其中49种蛋白（0.81%）在玻璃体腔 注射后表达上调超过5倍或下调超过80%。变化比较显著的蛋白主要与细胞骨架有关。显著变化蛋白富集的通路包括黏着斑 通路、紧密连接通路、核黄素新陈代谢通路、细胞外基质受体相互作用通路及肌动蛋白细胞骨架调节通路。功能网络图分析显 示ACTC1及ISG15在蛋白相互作用关系中起着比较中心的作用。结论玻璃体腔内注射PBS主要引起与细胞黏连、形态、迁 移、分化、信号传导及核黄素代谢通路相关的视网膜蛋白变化。

Abstract: Objective Intravitreal (IVT) injection has become one of the most commonly performed ophthalmologic procedures. We investigated the changes in retinal function and proteomics in rabbits receiving IVT injection of PBS to evaluate the safety of IVT injection. Methods Twenty Chinchilla rabbits were subjected to IVT injection of 50 μL PBS in the right eyes. On days 0, 4, 7 and day 14, the retinas of the rabbits were isolated after routine ophthalmic and electroretinogram examinations. The protein expressions in the retinas were quantified using tandem mass tag (TMT)-labeling coupled with LC-MS/MS, and bioinformatic analysis of the differentially expressed proteins (DEPs) was performed based on KEGG database to identify significantly enriched pathways. Functional network of the significant DEPs was analyzed using STRING. Results No noticeable fundus or functional changes occurred in the rabbit eyes following IVT injection of PBS. A total of 6042 retinal proteins were identified in the retina following the injection, among which 49 proteins (0.81%) exhibited over 5.0-fold up-regulation or over 80% downregulation relative to the control. Most of the distinctly up-regulated or down-regulated proteins were associated with the cytoskeleton. Significantly enriched pathways involved focal adhesion, tight junction, riboflavin metabolism, extracellular matrix-receptor interaction and regulation of actin cytoskeleton. Functional network analysis showed that ACTC1 and ISG15 played central roles in the protein interaction networks. Conclusion IVT PBS injection in rabbits causes alterations in proteins associated with cell adhesion, morphology, migration, differentiation, signal transduction and riboflavin metabolism, but the alterations of the retinal proteins appear not sufficient to cause observable pathology of the retina.