大鼠酒精性肝纤维化肝非实质细胞蛋白质组学研究

Proteome analysis of liver nonparenchymal cells from rats with alcoholic liver fibrosis

目的本文研究酒精对肝非实质细胞蛋白质表达的影响,以探讨酒精性肝纤维化的发病机制。方法大鼠酒精灌胃导致其发生肝纤维化。采用James染色法检测大鼠肝脏的病理学变化,通过Percoll密度梯度离心富集肝非实质细胞,再通过二维凝胶电泳(2DE)分离非实质细胞的蛋白质,经考马斯亮蓝(G250)染色,采用液相色谱串联质谱鉴定差异表达的蛋白质,并对部分差异蛋白质采用实时定量RT-PCR和免疫印迹的方法进行验证。对于2DE胶上的蛋白质点,采用两样本t检验的方法,对于RT-PCR分析,采用Mann-Whitney U检验进行统计分析。结果建立了酒精性肝纤维化大鼠模型,通过Percoll密度梯度离心纯化的非实质细胞中淋巴细胞、Kupffer细胞和内皮细胞分别富集了1.5、3.2和3.7倍。采用二维凝胶电泳法检测到了800多个蛋白质点,检测到具有2倍以上的差异蛋白质有26个,采用LC-MS法鉴定了21个非冗余蛋白质,对其中7个蛋白质的RT-PCR分析发现:ANXA3、CES3、ATPA和NDUFV2的mRNA水平和蛋白质组研究结果一致。结论本研究鉴定了一批与酒精性肝纤维化相关的蛋白质,可能为了解酒精性肝纤维化发病机制提供一些新线索。

Objective The development of alcoholic liver fibrosis （ALF）is a complex process involving both parenchymal and nonparen-chymal cells.The knowledge about the proteome of liver nonparenchymal cells （NPCs）in response to ethanol is limited.This paper aims to investigate the regulatory effect of alcohol on the protein expression in liver NPCs during liver fibrosis development and to provide new clues for understanding the molecular mechanism of liver fibrosis.Methods Rats were treated with ethanol by gastric administration to establish a liver fibrosis model.The pathological changes in the liver were evaluated by James staining.Liver NPCs were enriched by Percoll density gradient centrifugation,and proteins were extracted from NPCs by two -dimensional gel electrophoresis （2DE）and stained by Coomassie Brilliant Blue G -250.The differentially expressed proteins were identified by liquid chromatography -tandem mass spectrometry （LC -MS /MS）.Some of the differentially expressed proteins were verified by real time RT -PCR and Western blot.The protein spots on 2DE gels were analyzed by two sample t -test.The RT -PCR results were statistically analyzed by Mann -Whitney test.Results A rat model of ALF was established.Among the NPCs purified by Percoll density gradient centrifugation,lymphocytes,Kupffer cells,and endothelial cells were enriched for 1.5,3.2,and 3.7 times,respectively.More than 800 protein spots were detected by 2DE,and there were 26 proteins with more than 2 -fold increases or decreases in expression;21 non -redundant proteins were identified by LC -MS /MS and real -time RT-PCR analysis of 7 of these proteins showed that the mRNA levels of ANXA3,CES3,ATPA and NDUFV2 were in accordance with the pro-teome analysis results.Conclusion This study detected and identified a group of differentially expressed proteins related to ALF.Our work might offer some new clues to understand the mechanism of ALF.