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基于iTRAQ的大鼠穿透性角膜移植急性角膜移植排斥模型泪液定量蛋白质组学分析

iTRAQ-Based Quantitative Proteomic Analysis of Tear Fluid in a Rat Penetrating Keratoplasty Model With Acute Corneal Allograft Rejection.

作者信息

Huang Feifei, Xu Jianjiang, Jin Hong, Tan Jianwen, Zhang Chaoran

机构信息

Key Laboratory of Myopia Ministry of Health, Department of Ophthalmology, Eye, Ear, Nose and Throat Hospital, Fudan University, Shanghai, China.

Institutes of Biomedical Sciences, Fudan University, Shanghai, China.

出版信息

Invest Ophthalmol Vis Sci. 2015 Jun;56(6):4117-24. doi: 10.1167/iovs.14-16207.

Abstract

PURPOSE

This study aimed to develop a greater understanding of the mechanisms underlying acute corneal allograft rejection by identifying differentially expressed tear proteins at defined stages and discovering potentially important proteins involved in the process.

METHODS

The isobaric tags for relative and absolute quantitation (iTRAQ)-two-dimensional liquid chromatography-tandem mass spectrometry (2DLC-MS/MS) technique was used to identify tear proteins showing significant alterations in a rat penetrating keratoplasty model at different time points. Bioinformatics technology was applied to analyze the significant proteins, and a potential protein was verified by Western blotting.

RESULTS

A total of 269 proteins were quantified, and 118 proteins were considered to be significantly altered by at least 2.0- or 0.5-fold. For gene ontology annotations, the top enrichments were neurological disease, free radical scavenging, cell death and survival, and cell movement. For pathway analyses, the top enrichments were LXR/RXR activation, acute phase response signaling, clathrin-mediated endocytosis signaling, and coagulation system. Coronin-1A was verified as a potential protein involved in the early stage of acute corneal allograft rejection.

CONCLUSIONS

This study first demonstrates that tear proteomics is a powerful tool for better understanding of the mechanisms underlying acute corneal rejection, and that coronin-1A in tears might be closely related to allograft rejection.

摘要

目的

本研究旨在通过鉴定特定阶段差异表达的泪液蛋白,并发现该过程中潜在的重要蛋白,以更深入地了解急性角膜移植排斥反应的潜在机制。

方法

采用相对和绝对定量等压标签(iTRAQ)-二维液相色谱-串联质谱(2DLC-MS/MS)技术,鉴定大鼠穿透性角膜移植模型在不同时间点泪液蛋白的显著变化。应用生物信息学技术分析这些显著蛋白,并通过蛋白质印迹法验证一种潜在蛋白。

结果

共定量了269种蛋白,其中118种蛋白被认为有至少2.0倍或0.5倍的显著变化。基因本体注释显示,最富集的是神经疾病、自由基清除、细胞死亡与存活以及细胞运动。通路分析显示,最富集的是LXR/RXR激活、急性期反应信号传导、网格蛋白介导的内吞信号传导和凝血系统。冠蛋白-1A被验证为参与急性角膜移植排斥反应早期的一种潜在蛋白。

结论

本研究首次表明,泪液蛋白质组学是更好地理解急性角膜排斥反应机制的有力工具,且泪液中的冠蛋白-1A可能与移植排斥反应密切相关。

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