肝素治疗烧伤的蛋白质组学研究

Proteomic study on heparin treated burns

关键词:  肝素  烫伤  双向电泳  蛋白质组学  质谱分析

Key words:  heparin  burns  two-dimensional electrophoresis  proteomics  mass spectrum

摘要:

［目的］ 利用蛋白质组学的方法，寻找肝素处理烧伤后差异表达的蛋白质，进一步探索肝素治疗烫伤过程的相关蛋白质，以期阐明肝素治疗烫伤的机制。［方法］ 制作小鼠Ⅱ度烫伤模型，使用肝素外敷治疗，并在第3天从肝素组（烫伤后用肝素治疗）、烫伤组（烫伤后自然愈合）和对照组（未进行烫伤处理）中各取5只小鼠处死，提取烫伤及其周围皮肤组织的蛋白质，采用双向凝胶电泳（2-DE）技术分离各组的蛋白质，用考马斯亮蓝方法染色。通过凝胶成像系统获得双向电泳凝胶图谱后，用PD Quest图像分析软件比较分析，从而确定差异表达的蛋白质。选取差异最明显的蛋白质点，经胰蛋白酶水解后，通过基质辅助激光解吸附飞行时间串联质谱(MALDI-TOF-MS/MS)对其进行肽质谱分析并与数据库比对，鉴定待测蛋白质。［结果］ 烫伤组共检测出75个蛋白质点，肝素组共检测出95个蛋白质点，其中肝素处理后上调2倍以上的蛋白质点有10个，下调2倍以上的点有4个。质谱分析初步鉴定表达差异最大的蛋白质点为载脂蛋白A-I前体。［结论］ 烫伤创面经肝素处理后表现出不同的蛋白质表达谱。

Abstract:

［Objective］ To identify the differentially expressed proteins from burns after treatment with heparin for elucidation of the mechanism of heparin treatment on burns. ［Methods］ Second-degree burned mice were divided into three groups randomly: Heparin treated group (3000 U/mL heparin after burning), burned group (the wound without treatment) and control group (no burns). Three day after heparin treatment, the total proteins from the three groups were extracted and separated by two-dimensional gel electrophoresis (2-DE). The differentially expressed proteins were determined by PD Quest software and further identified with MALDI-TOF MS/MS. ［Results］ 2-DE PAGE profile revealed 95 protein spots from heparin-treated group and 75 spots from burned only group. Ten proteins were increased, and four proteins were decreased significantly after treated with heparin. The protein showing the highest difference was hydrolyzed with trypsin and identified as Apolipoprotein A-I precursor by mass spectrum analysis. ［Conclusion］ Some differentially expressed proteins from burns were revealed after treated with heparin.