梅花鹿致敏与休眠鹿茸干细胞差异蛋白表达的2D-DIGE分析
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- 英文论文题名：Analysis of Differentially Expressed Proteins in the Potentiated and the Dormant Antler Stem Cells through 2D-DIGE
- 论文作者：董振 ; 王权威 ; 刘振 ; 孙红梅 ; 李春义
- 英文论文作者：Dong Zhen ; Wang Quanwei ; Liu Zhen ; Sun Hongmei ; Li Chunyi ; State Key Lab for Molecular Biology of Special Animals ; Institute of Special Animal and Plant Sciences ; Chinese Academy of Agricultural Science
- 年：2016
- 作者机构：中国农业科学院特产研究所特种动物分子生物学国家重点实验室;
- 论文关键词：鹿茸干细胞 ; 再生 ; 蛋白质组学 ; 2D-DIGE
- 英文论文关键词：antler stem cell ; regeneration ; proteomics ; 2D-DIGE
- 会议召开时间：2016-06-01
- 会议录名称：“十二五” 鹿科学研究进展
- 语种：中文
- 分类号：S825
- 学会代码：ZXMY
- 学会名称：中国畜牧业协会
- 页数：17
- 文件大小：2030k
- 原文格式：O
摘要
【目的】对梅花鹿(Cervus nippon)致敏鹿茸干细胞与休眠鹿茸干细胞表达蛋白进行差异筛选、鉴定及生物信息分析,为深入探讨鹿茸独特的再生分子调节机制奠定基础。【方法】采用双向荧光差异凝胶电泳(Two-dimensional fluorescence difference in gel electrophoresis,2D-DIGE)分离蛋白样品;利用DeCyder 7.2分析软件对2D-DIGE图像进行统计学分析寻找差异表达蛋白;MALDI-TOF-MS(Matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry)鉴定差异蛋白,通过Mascot软件搜索NCBInr数据库寻找匹配的蛋白;采用PANTHER(Protein Analysis Through Evolutionary Relationships)软件对差异蛋白进行聚类分析,REACTOME数据库分析差异蛋白所参与的信号通路。【结果】得到了致敏鹿茸干细胞与休眠鹿茸干细胞2D-DIGE图谱,致敏鹿茸干细胞与休眠鹿茸干细胞蛋白丰度相比较比值≥1.1倍以及比值≤-1.1倍(P<0.05)的差异蛋白点有159个,其中110个上调表达,49个下调表达,EDA(Extended data analysis)分析得到了多个Marker蛋白,质谱鉴定了84个差异蛋白质点,48个为阳性结果,共来自27种蛋白质。并对已鉴定蛋白进行了GO分析以及信号通路富集分析。【结论】致敏鹿茸干细胞与休眠鹿茸干细胞蛋白差异明显,质谱鉴定获得了来自多种可能与鹿茸再生相关的差异蛋白。由此可知,鹿茸再生是鹿茸干细胞从休眠到致敏的转化过程,需要多种蛋白分子以及信号通路的综合调控。
【Objective】The objective of this study was to screen,identify and analyze the differentially expressed proteins in the potentiated and the dormant antler stem cells in sika deer(Cervus nippon),and then to shed new lights on the molecular mechanisms underlying antler regeneration.【Method】Differences in two- dimensional fluorescence of gel electrophoresis(2D-DICE) was utilized for the help to separate the protein spots;Differentially expressed protein spots were selected by DeCyder 2D(version 7.2);Matrix-assisted laser desorption/ionization time- of- flight tandem mass spectrometry(MALDI- TOF- MS) was carried out to obtain peptide mass fingerprinting,Mascot software was used to search against the NCBInr database;PANTHER(Protein Analysis Through Evolutionary Relationships) and REACTOME analysis were performed to further explore the involved signal pathways about these identified proteins.【Result】The proteomic profile of the potentiated antler stem cells compared with the dormant antler stem cells was explored by 2D- DICE.There were 159 protein spots with more than 1.1— fold changes and less than — 1.1— fold changes and P values less than 0.05 differentially expressed by the potentiated over the dormant antler stem cells,including 110 up- regulated and 49 down- regulated protein spots.Multiple markers were obtained by extended data analysis module.MALDI- TOF- MS identified 84 differentially expressed protein spots and 48 of them were positive results which came from 27 kinds of proteins.【Conclusion】There is a significant difference in proteomic level between the potentiated and the dormant antler stem cells,some proteins identified which are involved in multiple functional categories might be related to antler regeneration.Therefore,antler regeneration,which is regulated by multiple proteins and a complicated signal network,is a process from the dormant to the potentiated states in antler stem cells.
【Objective】The objective of this study was to screen,identify and analyze the differentially expressed proteins in the potentiated and the dormant antler stem cells in sika deer(Cervus nippon),and then to shed new lights on the molecular mechanisms underlying antler regeneration.【Method】Differences in two- dimensional fluorescence of gel electrophoresis(2D-DICE) was utilized for the help to separate the protein spots;Differentially expressed protein spots were selected by DeCyder 2D(version 7.2);Matrix-assisted laser desorption/ionization time- of- flight tandem mass spectrometry(MALDI- TOF- MS) was carried out to obtain peptide mass fingerprinting,Mascot software was used to search against the NCBInr database;PANTHER(Protein Analysis Through Evolutionary Relationships) and REACTOME analysis were performed to further explore the involved signal pathways about these identified proteins.【Result】The proteomic profile of the potentiated antler stem cells compared with the dormant antler stem cells was explored by 2D- DICE.There were 159 protein spots with more than 1.1— fold changes and less than — 1.1— fold changes and P values less than 0.05 differentially expressed by the potentiated over the dormant antler stem cells,including 110 up- regulated and 49 down- regulated protein spots.Multiple markers were obtained by extended data analysis module.MALDI- TOF- MS identified 84 differentially expressed protein spots and 48 of them were positive results which came from 27 kinds of proteins.【Conclusion】There is a significant difference in proteomic level between the potentiated and the dormant antler stem cells,some proteins identified which are involved in multiple functional categories might be related to antler regeneration.Therefore,antler regeneration,which is regulated by multiple proteins and a complicated signal network,is a process from the dormant to the potentiated states in antler stem cells.
引文
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[28]LOKHOV P G,MOSHKOVSKII S A,IPATOVA 0 M,et al.Cytosolic insulin-binding proteins of mouse liver cells[J],protein peptide lett,2004,11(1):29-33.
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[30]CLEVERS H,LOH K M,NUSSE R.An integral program for tissue renewal and regeneration;Wnt signaling and stem cell control[J].Science,2014,346(6205):1248012.
[31]MOUNT J G,MUZYLAK M,ALLEN S,et al.Evidence that the canonical Wnt signalling pathway regulates deer antler regeneration[J].dev dynam,2006,235(5):1 390-1 399.
[32]LIU L,CHOWDHURY S,FANG X,et al.Attenuation of unfolded protein response and apoptosis by mReg2 induced GRP78 in mouse insulinoma cells[J].febs letters,2014,588(11):2 016-2 024.
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[37]MERSFELDER E L.PARTHUN M R.The tale beyond the tail;Histone core domain modifications and the regulation of chromatin structure[J].Nucleic Acids Res,2006,34;2 653-2 662.
[38]KOUZARIDES T.Chromatin modifications and their function[J].Cell,2007,128(4):693-705.
[39]RUTHENBURG A J,ALLIS C D,WYSOCKA J.Methylation of lysine 4 on histone H3:intricacy of writing and reading a single epigenetic mark[J].mol cell,2007,25(1):15-30.
[40]MAKI N,TSONIS P A,AGATA K.Changes in global histone modifications during dedifferentiation in newt lens regeneration[J],mol vis,2010,16:1 893.
[41]ANCHELIM,MURCIA L,ALCARAZ-PEREZ F,et al.Behaviour of telomere and telomerase during aging and regeneration in zebrafish[J],plos one,2011,6(2):el6955.
[42]LUND T C,GLASS T J,TOLAR J,et al.Expression of telomerase and telomere length are unaffected by either age or limb regeneration in Danio rerio[J],plos one,2009,4(11):e7688.
[43]DI FAGAGNA F A,TEO S H,JACKSON S P.Functional links between telomeres and proteins of the DNA-damage response[J].Genes Dev.2004,18,1 781-1 799.
[44]KARRASCH S,WALKER J E.Novel features in the structure of bovine ATP synthase[J],j mol biol,1999,290:379-384.
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[50]SINGH S,SADACHARAN S,SU S,et al.Overexpression of vimentin;role in the invasive phenotype in an androgen-independent model of prostate cancer[J].Cancer Res,2003,63:2 306-2 311.
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[2]LI C,YANG F,SHEPPARD A.Adult stem cells and mammalian epimorphic regeneration-insights from studying annual renewal of deer antlers[J].Curr Stem Cell Res Ther,2009,4(3):237-251.
[3]LI C,PEARSON A,MCMAHON C.Morphogenetic mechanisms in the cyclic regeneration of hair follicles and deer antlers from stem cells[J].Biomed Res Int,2013,2013:643 601.
[4]UNSAL C,ORAN M,TURELI H 0,et al.Detection of subclinical atherosclerosis and diastolic dysfunction in patients with schizophrenia[J].Neuropsychiatr Dis Treat,2013,9;1 531-1 537.
[5]GARCIA M,CHARLTON BD,WYMAN M T,et al.Do Red Deer Stags(Cervus elaphus)Use Roar Fundamental Frequency(F0)to Assess Rivals[J].PLoS One,2013,8(12):e83946.
[6]CLARK DE,LI C,WANG W,et al.Vascular localization and proliferation in the growing tip of the deer antler[J].Anat Rec A Discov Mol Cell Evol Biol,2006,288(9):973-981.
[7]LI C.Deer antler regeneration:a stem cell-based epimorphic process[J].Birth Defects Res C Embryo Today,2012,96(1):51-62.
[8]KIERDORF U,KIERDORF H,SZUWART T.Deer antler regeneration;cells,concepts,and controversies[J].J Morphol,2007,268(8):726-738.
[9]LI C.Exploration of the mechanism underlying neogenesis and regeneration of postnatal mammalian skin-deer antler velvet[J].Int J Med Biol Front,2010,11/12(16):1-19.
[10]LIC,SUTTIE J M.Tissue collection methods for antler research[J].Eur J Morphol.2003,41(1):23-30.
[11]LI C,YANG F,LI G,et al.Antler regeneration;a dependent process of stem tissue primed via interaction with its enveloping skin[J].J Exp Zool A Ecol Genet Physiol,2007,307(2):95-105.
[12]PARK H J,LEE D H,PARK S G,et al.Proteome analysis of red deer antlers[J].Proteomics,2004,4(11):3 642-3 653.
[13]PRICE J,FAUCHEUX C,ALLEN S.Deer antlers as a model of Mammalian regeneration[J].Curr Top Dev Biol,2005,67:1-48.
[14]LI C,HARPER A,PUDDICK J,et al.Proteomes and signalling pathways of antler stem cells[J],plosone,2012,7(1):e30026.
[15]丁新伦,谢荔岩,吴祖建.水稻草状矮化病毒侵染寄主水稻差异表达蛋白的鉴定和分析[J].中国农业科学,2014,47(9):1 725-1 734.DING X,XIE L,WU Z.Identification and Analysis of Differentially Expressed Proteins of Host Rice(Oryza sativa)Infected with Rice Grassy Stunt Virus[J].Scientia Agriculture Sinica.2014,47(9):1725-1734.(in Chinese)
[16]曹晓艳,冯建荣,王大江,白茹,刘月霞.2D-DIGE技术研究自交不亲和杏品种‘新世纪'花柱表达蛋白[J].中国农业科学,2011,44(4):789-797.CAO X,FENG J,WANG D,et al.Reaserch of Protein Expression of Style In Sell-Incompatibility Cultivar Prunus armeniaca L.cv.Xinshiji by 2D-DIGE Technique[J]-Scientia Agricultura Sinica.2011,44(4):789-797.(in Chinese)
[17]MI H,MURUGANUJAN A,THOMAS PD.PANTHER in 2013:modeling the evolution of gene function,and other gene attributes,in the context of phylogenetic trees[J].Nucleic Acids Res,2013,41(Dl):D377-D386.
[18]HUANG D W,SHERMAN B T,LEMPICKI R A.Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources[J].Nat Protoc,2009,4;44-57.
[19]HUANG DW,SHERMAN B T,LEMPICKI R A.Bioinformatics enrichment tools:paths toward the comprehensive functional analysis of large gene lists[J].Nucleic Acids Res,2009,37;1-13.
[20]BERA T K,SAINT FLEUR A,LEE Y,et al.POTE paralogs are induced and differentially expressed in many cancers[J].CANCER RES,2006,66(1):52-56.
[21]BERA T K,FLEUR A S,HA D,et al.Selective POTE paralogs on chromosome 2 are expressed in human embryonic stem cells[J].STEM CELLS DEV,2008,17(2):325-332.
[22]KESSLER J D,KAHLE K T,SUN T.A SUMOylation-dependent transcriptional subprogram is required for Myc-driven tumorigenesis[J].Science,2012,335(6066):348-353.
[23]AMENTE S,LAVADERA M L,DI PALO G,et al.SUMO-activating SAE1 transcription is positively regulated by Myc[J].AM J CANCER RES,2012,2(3):330.
[24]EVAN G.Taking a backdoor to target Myc[J].Science,2012,335(6066):293-294.
[25]SUMI T,TSUNEYOSHI N,NAKATSUJI N,et al.Apoptosis and differentiation of human embryonic stem cells induced by sustained activation of c-Myc[J].Oncogene,2007,26(38):5 564-5 576.
[26]STEGER V,MOLNAR A,BORSY A.Antler development and coupled osteoporosis in the skeleton of red deer Cervus elaphus;expression dynamics for regulatory and effector genes[J].MOL GENET GENOMICS,2010,284(4):273-287.
[27]WANG Y,KURAMITSU Y,TAKASHIMA M.Identification of four isoforms of aldolase B downregulated in hepatocellular carcinoma tissues by means of two-dimensional Western blotting[J].In Vivo,2011,25(6):881-886.
[28]LOKHOV P G,MOSHKOVSKII S A,IPATOVA 0 M,et al.Cytosolic insulin-binding proteins of mouse liver cells[J],protein peptide lett,2004,11(1):29-33.
[29]CASPI M,PERRY G,SKALKA N,et al.Aldolase positively regulates of the canonical Wnt signaling pathway[J].Mol cancer,2014,13(1):164.
[30]CLEVERS H,LOH K M,NUSSE R.An integral program for tissue renewal and regeneration;Wnt signaling and stem cell control[J].Science,2014,346(6205):1248012.
[31]MOUNT J G,MUZYLAK M,ALLEN S,et al.Evidence that the canonical Wnt signalling pathway regulates deer antler regeneration[J].dev dynam,2006,235(5):1 390-1 399.
[32]LIU L,CHOWDHURY S,FANG X,et al.Attenuation of unfolded protein response and apoptosis by mReg2 induced GRP78 in mouse insulinoma cells[J].febs letters,2014,588(11):2 016-2 024.
[33]PFAFFENBACH K T,PONG M,MORGAN T E,et al.GRP78/BiP is a novel downstream target of IGF-1 receptor mediated signaling[J],j cell physiol,2012,227(12):3 803-3 811.
[34]PFAFFENBACH K T,LEE A S.The critical role of GRP78 in physiologic and pathologic stress[J],curr opin cell biol,2011,23(2):150-156.
[35]PARYS J B,DECUYPERE J P,BULTYNCK G.Role of the inositol 1,4,5-trisphosphate receptor/Ca 2+-release channel in autophagy[J].Cell Commun Signal,2012,10;17.
[36]TANIMURA S,HIRAN A I,HASHIZUME J,et al.Anticancer drugs up-regulate HspBPl and thereby antagonize the prosurvival function of Hsp70 in tumor cell[J].j biol chem,2007,282(49):35430-35439.
[37]MERSFELDER E L.PARTHUN M R.The tale beyond the tail;Histone core domain modifications and the regulation of chromatin structure[J].Nucleic Acids Res,2006,34;2 653-2 662.
[38]KOUZARIDES T.Chromatin modifications and their function[J].Cell,2007,128(4):693-705.
[39]RUTHENBURG A J,ALLIS C D,WYSOCKA J.Methylation of lysine 4 on histone H3:intricacy of writing and reading a single epigenetic mark[J].mol cell,2007,25(1):15-30.
[40]MAKI N,TSONIS P A,AGATA K.Changes in global histone modifications during dedifferentiation in newt lens regeneration[J],mol vis,2010,16:1 893.
[41]ANCHELIM,MURCIA L,ALCARAZ-PEREZ F,et al.Behaviour of telomere and telomerase during aging and regeneration in zebrafish[J],plos one,2011,6(2):el6955.
[42]LUND T C,GLASS T J,TOLAR J,et al.Expression of telomerase and telomere length are unaffected by either age or limb regeneration in Danio rerio[J],plos one,2009,4(11):e7688.
[43]DI FAGAGNA F A,TEO S H,JACKSON S P.Functional links between telomeres and proteins of the DNA-damage response[J].Genes Dev.2004,18,1 781-1 799.
[44]KARRASCH S,WALKER J E.Novel features in the structure of bovine ATP synthase[J],j mol biol,1999,290:379-384.
[45]IZQUIERDO JM.Control of the ATP synthase beta subunit expression by RNA-binding proteins TIA-1,TIAR,and HuR[J].Biochem Biophys Res Commun,2006,348:703-711.
[46]ERIKSSON J E,DECHAT T,GRIN B,et al.Introducing intermediate filaments:from discovery to disease[J].j clin invest,2009,119:1763-1771.
[47]ECKES B,COLUCCI-GUYON E,SMOLA H,et al.Impaired wound healing in embryonic and adult mice lacking vimentin[J],j cell sci,2000,113(13):2455-2462.
[48]ECKES B,DOGIC D,COLUCCI-GUYON E.Impaired mechanical stability,migration and contractile capacity in vimentin-deficient fibroblasts[J].j cell sci,1998,111:1 897-1 907.
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