荧光双向电泳检测雌雄小鼠肝组织蛋白组学的差异
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摘要
目的检测雌雄小鼠肝组织蛋白质组表达差异,探讨肝病发生的性别差异机制。方法采用双向荧光差异凝胶电泳(two dimension difference gel electrophoresis,2D-DIGE)和基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ionization time of flight mass spectrometry,MALDI-TOF-MS)技术检测C57BL/6J雌雄小鼠肝组织的蛋白质组差异表达谱,分离并鉴定差异表达蛋白,应用Western blot进行验证,并对差异表达蛋白进行生物信息学分析,包括蛋白功能注释、分类分析、京都基因与基因组百科全书通路分析。结果经双向荧光差异凝胶电泳-图像分析得到1767个蛋白点,其中差异倍数≥1.5(P<0.05)的蛋白点325个。从中选择78个差异蛋白点进行MALDI-TOF-MS鉴定,得到48种差异性表达的蛋白质。其中,与雌鼠相比,雄鼠肝组织中高表达的蛋白有14种,低表达的蛋白有34种。选取6个差异蛋白点进行Western blot验证,证明了2D-DIGE结果的可靠性。GO分析结果显示,涉及雌雄鼠肝组织中的差异蛋白在细胞组分、分子功能、生物学过程中的分布广泛。KEGG通路分析发现差异蛋白涉及6条信号通路。结论 C57BL/6J雌雄小鼠肝组织的差异性蛋白表达的检测结果为研究性别差异性肝病发生的分子机制提供了基础生物学信息和有益的线索。
Objective To identify the differential proteomic expressions between the liver tissues of male and female mice,and investigate the mechanisms underlying gender differences in liver diseases. Methods Two-dimensional fluorescence difference gel electrophoresis( 2D-DIGE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry( MALDI-TOF-MS) were used to identify the differentially expressed proteins in the liver tissues of male and female C57BL/6J mice. The differentially expressed proteins were validated by Western blot and further analyzed by bioinformatics,including Gene Ontology( GO) analysis and Kyoto Encyclopedia of Genes and Genomes( KEGG). Results Among the auto-detected 1767 protein spots by 2D-DIGE,325 protein spots were differentially expressed( | ratio | ≥1. 5,P< 0. 05) between the liver tissues of male and female mice,in which 78 spots were randomly selected for MALDI-TOF-MS identification and finally 48 distinct proteins were obtained. Compared with females,14 and 34 proteins were up-or downregulated in males,respectively. Among them,6 differentially expressed proteins were validated by Western blot whichconfirmed the reliability of 2D-DIGE results. GO analysis showed that the differentially expressed proteins in the liver tissues of male and female mice are associated to various cellular component,molecular function and biological process. 6pathways were significantly different between the liver tissues of males and females depending on KEGG analysis.Conclusions The proteomic data and related analysis of the liver tissues of C57BL/6J mice offer crucial clues for elucidating the underlying mechanisms of different gender effects on liver diseases.
Objective To identify the differential proteomic expressions between the liver tissues of male and female mice,and investigate the mechanisms underlying gender differences in liver diseases. Methods Two-dimensional fluorescence difference gel electrophoresis( 2D-DIGE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry( MALDI-TOF-MS) were used to identify the differentially expressed proteins in the liver tissues of male and female C57BL/6J mice. The differentially expressed proteins were validated by Western blot and further analyzed by bioinformatics,including Gene Ontology( GO) analysis and Kyoto Encyclopedia of Genes and Genomes( KEGG). Results Among the auto-detected 1767 protein spots by 2D-DIGE,325 protein spots were differentially expressed( | ratio | ≥1. 5,P< 0. 05) between the liver tissues of male and female mice,in which 78 spots were randomly selected for MALDI-TOF-MS identification and finally 48 distinct proteins were obtained. Compared with females,14 and 34 proteins were up-or downregulated in males,respectively. Among them,6 differentially expressed proteins were validated by Western blot whichconfirmed the reliability of 2D-DIGE results. GO analysis showed that the differentially expressed proteins in the liver tissues of male and female mice are associated to various cellular component,molecular function and biological process. 6pathways were significantly different between the liver tissues of males and females depending on KEGG analysis.Conclusions The proteomic data and related analysis of the liver tissues of C57BL/6J mice offer crucial clues for elucidating the underlying mechanisms of different gender effects on liver diseases.
引文
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[2]Pan JJ,Fallon MB.Gender and racial differences in nonalcoholic fatty liver disease[J].World J Hepatol,2014,6(5):274-283.
[3]Wang AG,Moon HB,Lee MR,et al.Gender-dependent hepatic alterations in H-ras12V transgenic mice[J].J Hepatol,2005,43(5):836-844.
[4]定明,何允刚,张小飞,等.蛋白质组学技术筛选和鉴定白化黑线仓鼠皮肤白化相关差异蛋白质[J].中国实验动物学报,2014,22(1):79-82.
[5]李顺,陈丽香,彭秀华,等.小鼠肝癌模型研究进展[J].中国实验动物学报,2016,24(2):213-216.
[6]Boguski MS,Mc Intosh MW.Biomedical informatics for proteomics[J].Nature,2003,422(6928):233-237.
[7]Holbrook NJ,Ikeyama S.Age-related decline in cellular response to oxidative stress:links to growth factor signaling pathways with common defects[J].Biochem Pharmacol,2002,64(5-6):999-1005.
[8]Thu VT,Kim HK,Ha SH,et al.Glutathione peroxidase 1protects mitochondria against hypoxia/reoxygenation damage in mouse hearts[J].Pflugers Arch,2010,460(1):55-68.
[9]Shibata S,Shibata N,Shibata T,et al.The role of Prdx6 in the protection of cells of the crystalline lens from oxidative stress induced by UV exposure[J].Jpn J Ophthalmol,2016,60(5):408-418.
[10]Jadeja RN,Urrunaga NH,Ahmad D,et al.Data regarding M1muscarinic receptor-mediated modulation of hepatic catalase activity in response to oxidative stress[J].Data Brief,2016,6:405-409.
[11]Cohen JC,Horton JD,Hobbs HH.Human fatty liver disease:old questions and new insights[J].Science,2011,332(6037):1519-1523.
[12]Lu X,Yuan ZY,Yan XJ,et al.Effects of Angelica dahurica on obesity and fatty liver in mice[J].Chin J Nat Med,2016,14(9):641-652.
[13]Fernandez-Miranda C,Perez-Carreras M,Colina F,et al.A pilot trial of fenofibrate for the treatment of non-alcoholic fatty liver disease[J].Dig Liver Dis,2008,40(3):200-205.
[14]周玲丽,曹骥,李薇,等.RNA干扰FABP5基因对人肝癌Hep G2细胞裸鼠移植瘤生长的影响[J].中国病理生理杂志,2015,31(4):603-608.
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