肝再生早期线粒体差异表达蛋白质组分析

Differential proteomic analysis of mitochondria during the early phase of liver regeneration after partial hepatectomy

目的探讨线粒体在肝再生早期的作用,寻找肝再生早期的标志蛋白。方法 30只SD大鼠随机均分为70%肝部分切除组(PH组)和假手术组(SH组),每组再分为3个小组,每组5只,手术24h后处死大鼠,取相同部位的肝组织进行检测。PH组和SH组配对,独立重复实验3次;Nycodenz密度梯度离心分离线粒体,二维电泳(2-DE)分离线粒体蛋白质,采用PDQuest 7.4软件分析2-DE图谱;MALDI-TOF MS技术鉴定差异表达蛋白,采用Mascot方法进行数据检索,对部分差异蛋白进行Western blotting验证。结果共筛选出25个差异表达蛋白点(P<0.05);与SH组比较,PH组中有3个蛋白点上调,22个蛋白点下调。质谱技术成功鉴定出其中22个差异蛋白。生物信息学分析显示这些差异蛋白参与了多条代谢途径,如糖代谢、脂代谢、呼吸链和氧化磷酸化、生物转化以及信号转导等。结论肝再生早期,线粒体通过多条代谢途径对其进行调节;亚细胞蛋白质组学技术有利于寻找肝再生相关的标志蛋白,为肝再生的深入研究提供新的方向和思路。

Objective To explore mitochondrial proteins correlated with the early phase of rat liver regeneration （LR） after partial hepatectomy （PH）, in order to better understand the role of mitochondria during LR through subcellular proteomics analysis. Methods The hepatectomy model was established by 70% hepatectomy （PH group）, and the sham operated rats （SH group） underwent the same surgical protocol without hepateetomy. Thirty SD rats were randomly divided into these two groups and all experiments were done in triplicate （n= 5）. Rats were sacrificed at 24h after operation and the hepatic tissue in similar parts were harvested. A purified mitochondrial fraction was obtained by Nycodenz density gradient centrifugation. The mitochondrial protein was extracted and analyzed by two dimensional gel electrophoresis. The protein spots on PH and SH gels were analyzed by PDQuestT. 4 software and MALDI-TOF mass spectrometric analysis. Mascot was used for the identification of proteins. The differential proteins were then validated by Western blotting. Results A total of 25 protein spots （P〈0.05） were identified, of which 3 proteins were up-regulated and 22 proteins were down-regulated in PH group compared with the SH group. Twenty two differentially expressed proteins were successfully identified by MALDI-TOF MS analysis. Analysis by bioinformatics showed that these differential proteins were associated with carbohydrate metabolism, lipid metabolism, respiratory chain and oxidation phosphorylation, biotransformation and signaling transduction. Conclusions It is revealed by the present study that hepatic mitoehondrial adaptations to liver regeneration after 70% hepatectomy may affect various cellular metabolic pathways. The subcellular proteomics may contribute greatly to looking for the biomarkers during liver regeneration, and offer a new method and direction for the further research in liver regeneration.