兔脑死亡状态对肝脏蛋白质表达谱的影响

摘要

目的 分析兔脑死亡状态下肝脏存在的差异蛋白质,为揭示脑死亡状态下肝脏损伤的影响因素提供实验依据.方法 采用缓慢颅内加压法建立兔脑死亡模型.提取脑死亡后6h标本蛋白质进行双向凝胶电泳.利用PDQuest凝胶图像分析软件对图像进行分析.将两组间差异在2倍以上的蛋白质点行基质辅助激光解析离子化飞行时间质谱鉴定.在NCBI数据库中检索,鉴定出相应的蛋白质,并用蛋白印迹法进行验证.结果 双向凝胶电泳显示,假手术组可检测到大约(973±34)个蛋白质点,脑死亡组可检测到大约(987±38)个蛋白质点.经成组比较共计有52个明显差异表达的蛋白质点,与假手术组相比,上调29个,下调23个.10个差异蛋白点分别为线粒体醛脱氢酶、过氧化物酶6、3磷酸肌醇依赖性蛋白激酶1、3-巯基丙酮酸硫基转移酶、乙醇脱氢酶、二氢嘧啶酶相关蛋白4、Runt相关转录因子1、无机焦磷酸酶、谷氨酸-半胱氨酸连接酶的调节亚基、微粒细胞色素B5.其中,RUNX1是我们比较关注的一个蛋白.通过蛋白印迹法对RUNX1在各组织中的表达情况进行验证发现,随着脑死亡时间的延长,RUNX1在肝脏中的表达逐渐减少.结论 双向凝胶电泳结合质谱鉴定是差异蛋白质组学研究的可靠平台和有力工具,鉴定出的蛋白质RUNX1可能与脑死亡后肝脏损伤的发生、发展有关,有助于对脑死亡后肝脏损伤机制的了解.%Objective To explore the differential proteins in livers with the help of proteomics,which provide experimental basis for the study of influence factors of liver injury in the state of brain-death.Methods Slow intracranial pressure method was used to establish the rabbit brain death model.Each liver tissue from 6 h after brain death of rabbit was collected.Total proteins were extracted and separated by two-dimensional gel electrophoresis.The image was analyzed by PDQuest software.The differentially expressed proteins between the two groups in more than two-fold were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry and retrieved in the NCBI database to identify the corresponding protein.And the different proteins were re-identified by western blot.Results Two-dimensional gel electrophoresis showed that there were about 973 ± 34 and 987 ± 38 protein spots in sham and brain death groups.A total of 52 differentially expressed protein spots between the two groups,29 were up-regulated,and 23 were down-regulated.10 different proteins were:DPYL4,ALDH2,PRDX6,PDK1,THTM,RUNX1,PPA1,ADH,GCLR,CYB5.RUNX1 is a protein of interest,so the expression of RUNX1 was detected by western blot and it showed that the expression of RUNX1 in liver decreased gradually in a time-dependent manner.Conclusions Two-dimensional gel electrophoresis and mass spectrometry identification is a reliable platform and powerful tool for differential proteomics studies.Identified protein RUNX1 may be related with liver injury after brain death,which is beneficial for the understanding of the mechanism of liver damage after brain death.