神经源性休克死亡家兔心肌组织差异蛋白分析
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摘要
目的
建立家兔神经源性休克死亡模型,运用比较蛋白质组学筛选神经源性休克死亡组与正常对照组表达差异的蛋白质,发现潜在的神经源性休克生物标记物,为诊断神经源性休克死亡提供客观依据,探索神经源性休克死亡的机制。
方法
1.采用外力打击家兔颈动脉窦建立神经源性休克死亡模型。
2.采用双向凝胶电泳技术分离神经源性休克死亡组及正常对照组左心室全蛋白,用PDQUEST8.0软件分析双向凝胶电泳图谱,筛选出表达显著差异的蛋白点,胰酶酶解后,用HPLC-Chip/MS进行在线分析、鉴定出差异表达蛋白质。
3.利用Mascot软件在UniProtKB/SWISS-PORT数据库中筛选差异表达蛋白质,进行生物信息学分析,获取差异蛋白质相关肽段的MS/MS质谱图。
结果
1.成功验证了前期研究中神经源性休克死亡的动物模型。
2.筛选出8个神经源性休克死亡组表达上调的蛋白质,分别是:角蛋白10、角蛋白15、α-2B肾上腺素能受体、线粒体过氧化物歧化酶、过氧化物酶增殖物激活受体δ、封闭蛋白1、锌指蛋白569、热休克蛋白70,3个表达下调的蛋白质,分别是:甲基转移酶4、膜联蛋白XIIIb、组织因子途径抑制物。
结论
家兔神经源性休克死亡发生了蛋白表达谱的改变,鉴定出的11种蛋白是潜在的神经源性休克生物标记物,可成为神经源性休克死亡诊断的客观依据。
Objectives
To establish neurogenic shock death model of animal. Analysis of proteins expressed differentially between neurogenic shock death group and normal control group by comparative proteomics analysis,and provide new experimental evidences to further elucidate the death mechanism of neurogenic shock from proteomic level. In order to find latent biomarkers of neurogenic shock and to offer essential diagnosticate datas of neurogenic shock death.
Methods
1.To establish neurogenic shock death model by hitting on the carotid sinus in rabbits.
2. We set apart the proteins in left ventricular in neurogenic shock death group and normal control group by 2-DE.The 2-DE images were analyzed with PDQuest8.0 software. Trypsin digested differentia protein spots selected. HPLC-Chip/MS was adopted to on-line analyze and indentify proteins expressed differentially.
3. Mascot software was used to search for the proteins expressed differentially in UniProtKB/SWISS-PORT, bioinformatics analysis can gain MS/MS spectrum of peptide from differentially expressed proteins.
Results
1.Successfully verified neurogenic shock death model of animal in our previous study.
2. 8 proteins upregulated significantly in established neurogenic shock death group were identified, including keratin 10、alpha-2B adrenergic receptor、superoxide dismutase, mitochondrial precursor、peroxisome proliferator activated receptor delta、zinc finger protein -569、hsc70-interacting protein、keratin 15、claudin 1,and as well as 3 proteins downregulated significantly including methyltransferase-like protein4、annexinXIIIb、tissue factor pathway inhibitor precursor.
Conclusions
The proteins expression have changed in neurogenic shock death group .The proteins which were identified are latent biomarkers and can offer possible diagnosticate datas of neurogenic shock death.
建立家兔神经源性休克死亡模型,运用比较蛋白质组学筛选神经源性休克死亡组与正常对照组表达差异的蛋白质,发现潜在的神经源性休克生物标记物,为诊断神经源性休克死亡提供客观依据,探索神经源性休克死亡的机制。
方法
1.采用外力打击家兔颈动脉窦建立神经源性休克死亡模型。
2.采用双向凝胶电泳技术分离神经源性休克死亡组及正常对照组左心室全蛋白,用PDQUEST8.0软件分析双向凝胶电泳图谱,筛选出表达显著差异的蛋白点,胰酶酶解后,用HPLC-Chip/MS进行在线分析、鉴定出差异表达蛋白质。
3.利用Mascot软件在UniProtKB/SWISS-PORT数据库中筛选差异表达蛋白质,进行生物信息学分析,获取差异蛋白质相关肽段的MS/MS质谱图。
结果
1.成功验证了前期研究中神经源性休克死亡的动物模型。
2.筛选出8个神经源性休克死亡组表达上调的蛋白质,分别是:角蛋白10、角蛋白15、α-2B肾上腺素能受体、线粒体过氧化物歧化酶、过氧化物酶增殖物激活受体δ、封闭蛋白1、锌指蛋白569、热休克蛋白70,3个表达下调的蛋白质,分别是:甲基转移酶4、膜联蛋白XIIIb、组织因子途径抑制物。
结论
家兔神经源性休克死亡发生了蛋白表达谱的改变,鉴定出的11种蛋白是潜在的神经源性休克生物标记物,可成为神经源性休克死亡诊断的客观依据。
Objectives
To establish neurogenic shock death model of animal. Analysis of proteins expressed differentially between neurogenic shock death group and normal control group by comparative proteomics analysis,and provide new experimental evidences to further elucidate the death mechanism of neurogenic shock from proteomic level. In order to find latent biomarkers of neurogenic shock and to offer essential diagnosticate datas of neurogenic shock death.
Methods
1.To establish neurogenic shock death model by hitting on the carotid sinus in rabbits.
2. We set apart the proteins in left ventricular in neurogenic shock death group and normal control group by 2-DE.The 2-DE images were analyzed with PDQuest8.0 software. Trypsin digested differentia protein spots selected. HPLC-Chip/MS was adopted to on-line analyze and indentify proteins expressed differentially.
3. Mascot software was used to search for the proteins expressed differentially in UniProtKB/SWISS-PORT, bioinformatics analysis can gain MS/MS spectrum of peptide from differentially expressed proteins.
Results
1.Successfully verified neurogenic shock death model of animal in our previous study.
2. 8 proteins upregulated significantly in established neurogenic shock death group were identified, including keratin 10、alpha-2B adrenergic receptor、superoxide dismutase, mitochondrial precursor、peroxisome proliferator activated receptor delta、zinc finger protein -569、hsc70-interacting protein、keratin 15、claudin 1,and as well as 3 proteins downregulated significantly including methyltransferase-like protein4、annexinXIIIb、tissue factor pathway inhibitor precursor.
Conclusions
The proteins expression have changed in neurogenic shock death group .The proteins which were identified are latent biomarkers and can offer possible diagnosticate datas of neurogenic shock death.
引文
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[2] Gordon I, Shapiro H A .Forensic Medicine-a guide to principles.(2nd ed)[M].Edinburgh Livingston,1982,57(60):10,13-16.
[3]高昌启,张晓艳,吴玉莲等.刺激颈动脉窦致死1例[J].刑事技术,2003, 1:53.
[4] Bold AJ,Borenstein HB, Veress AT, et al. A rapid and potent natriuretic response to intravenous injection of atrial myocardial extract in rats[J]. Life Sci,1981,28:89-94.
[5]李三强,崔旭红,马灵筠等.热休克蛋白70研究的现状及展望[J].中华实用中西医杂志,2009,2(4):246-247.
[6] Mestril R, Chi SH, Sayen MR, et al. Expression of inducible stress protein 70 in rat heart myogenic Cells confers protection against simulated ischemia-induced injury[J].J Clin Invest 1994,93(2):759-767.
[7] Heads RJ,Latchman DS,Yellon DM.Stable high level expression of a transfected human HSP70 gene protects a heart-derived muscle Cell line against thermal stress[J].Mol Cell Cardiol 1994;26(6):695-699.
[8] Robson Q, Alireza R. Ixolaris binding to factor X reveals a precursor state of factor Xa heparin-binding exosite[J]. Protein Science ,2008, 17:146–153.
[9] Brodde OE, Bruck H, Leineweber K.Cardiac Adrenoceptors: Physiological and Pathophysiological Relevance [J]. PharmacolSci,2006,100:323-337.
[10] Gavras I, Manolis AJ,Gavras H. Theα2-adrenergic receptor in hypertension and heart failure:experimental and clinical studies[J].Hypertens,2001,19:2115-2124.
[11] Mitic L L, Anderson J M. Molecular architecture of tight junctions[J]. Annual Rev Physiology, 1998, 60: 121-142.
[12] Inai T, Kobayashi J, Shibata Y. Claudin-1 contributes to the epithelial barrier function in MDCK cells[J]. Eur J Cell Biol,1999; 78:849–55.
[13]赵克森,黄巧冰.血管通透性增高的基本机制[J].中国病理生理杂志.2003,04:0549-0505.
[14]冼励坚.维生素B2在减低ADR毒性时对血清同功酶及心肌病变影响的实验研究[J].癌症,1984,2(3):94.
[15] Higashiyama H, Billin AN, Okamoto Y. Expression profiling of peroxisome proliferator-activated receptor-delta(PPAR-delta) in mouse tissues using tissue microarray[J]. Histochem Cell Biol,2007, 127: 485-494.
[16] Cheng L, Ding G, Qin Q. Cardiomyocyte-restricted peroxisome proliferator- activated receptor-delta deletion perturbs myocardial fattyacid oxidation and leads to cardiomyopathy[J]. Nat Med. 2004, 10: 1245-1250.
[17] Chang L ,Karin M.Mammalian MAP kinase signaling cascades [J].Nature.2001,410:37-40.
[18]黄欣琼,朱传炳,王跃群等.人类基因ZNF569结构域在MAPK信号途径中的作用.湖南师范大学自然科学学报[J].2005,28(3):80-84.
[19] DAVIS R J.Signal transduction by the.INK group of MAP kinases[J].Cell, 2000,103:239-252.
[20] Lafont F, Lecat S, Verkade P. Annexin XIIIb associates with lipid microdomains to function in apical delivery [J]. Cell Biol,1998,142:1413-1427.
[21] Lecat S, Verkade P, Thiele C. Different properties of two isoforms of annexin XIII in MDCK cells[J]. Journal of Cell Science ,2000,113:2607-2618 .
[22] Ohkawa T, Naomoto Y,Takaoka M, et al. Localization of heparanase in esophageal cancer cells: respective roles in prognosis and differentiation[J]. Lab Invest, 2004,84: 1289-1304.
[23] Kuo T. Cytokeratin profiles of the thymus and thymomas: histogenetic correlations and proposal for a histological classification of thymomas[J].Histopathology, 2000,36:403 -414.
[24] Zhao HW, Li LJ, Xie WY. Clinic significance of morphometric study on squamous cell carcinoma of floor of mouth. Hua Xi Kou Qiang Yi Xue Za Zhi, 2004,22:220-223.
[25] Maeda T,Takenaka K,Taguchi K, et al. Adenosquamous carcinoma of the liver:clinicopathologic characteristics and cytokeratin profile[J]. Cancer, 1997, 80:364-371.
[26] Lyle S,Christofidou-Solomidou M ,Liu YP,et a1.The C8/144B monoclona1 antibody recognizes cytokeratin 15 and defines the location of human hair follicle stem cells[J]. Cell Sci,1998,111(Pt 21):3179-3188.
[1] Wi1kins MR,Sanchez JC,Gooley AA.Progress with proteome projects :why all proteins expressed by a genome should be identified and how to do it[J].Biotechnol Genet Eng Rev,1996,13:19—27.
[2] Blackstock W ,Mann M.A boundless future for proteomics [J].TrendsBiotechnol,2001,19(Supp):s1-s2.
[3] Nyman TA.The role of mass spectrometry in preteome studies[J].Biomolecular Engineering,2001,18 :221-227.
[4] Yates JR 3rd. Mass spectrometry from genomics to proteomics[J].Trends Genet,2000,16 :5-8.
[5] Larsen M R,Larsen P M,Fey S J,et a1.Characterization of differently processed forms of enolase 2 from Saccharomyces cerevisiae by two-dimensional gel electrophoresis and mass spectrometry [J].Electrophoresis,2001,22(3):566—575.
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