吗啡耐受大鼠脊髓蛋白质组双向电泳图谱的建立与差异蛋白鉴定
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摘要
目的:建立吗啡耐受大鼠腰段脊髓组织蛋白质组双向凝胶电泳(two-dimensional gel electrophoresis,2-DE)图谱,比较分析吗啡耐受大鼠腰段脊髓组织中蛋白质组的变化和差异表达,鉴定出差异表达的蛋白质点并进行验证。方法:将16只鞘内置管雄性SD大鼠随机分为吗啡耐受组(MT组,n=8)和生理盐水组(NS组,n=8),取其腰段脊髓组织蛋白以固相pH梯度等电聚焦(immobilized pH gradients isoelectric focusing,IPGIEF)为第一向,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electroporesis,SDS-PAGE)为第二向进行2-DE,应用PDQuest分析软件对考马斯亮蓝染色的2-DE图谱进行图像分析,对差异蛋白质点进行基质辅助激光解吸电离飞行时间质谱(matrixassisted laser desorption/ionization time of flight mass spectrometry,MALDI-TOF-MS)分析,并利用Mascot查询软件搜索SwissProt数据库进行生物信息学分析。采用蛋白质印迹法验证部分差异蛋白。结果:MT组和NS组大鼠脊髓组织2-DE图谱中各分离出约1 000个清晰的蛋白质点,其中明显差异表达的蛋白有36个。采用MALDI-TOF-MS分析,并利用Mascot查询软件搜索Swiss-Prot数据库,共搜索到14个蛋白质,与NS组比较,MT组中表达下调的蛋白质点有2个,表达上调的蛋白点有12个。采用蛋白质印迹法对其中的NADH脱氢酶及伽玛烯醇化酶蛋白质点进行验证,结果与蛋白质组学结果一致。结论:初步建立了吗啡耐受大鼠脊髓组织蛋白质组双向电泳图谱,证明吗啡耐受可以引起脊髓中多种蛋白质的表达改变。
Objective:To establish a two-dimensional gel electrophoresis(2-DE) map for comparative proteomic analysis of rat spinal cord with chronic morphine tolerance,and to detect differentially expression proteins that are associated with chronic morphine tolerance.Methods:Sixteen male SD rats received the intrathecal catheterization operation and they were randomly divided into a morphine tolerance group(MT group,n=8) and a saline group(NS group,n=8).The lumbar enlargement segments of the MT group and the NS group spinal cord were harvested and proteins were separated by 2-DE.Differential proteome profiles were established and analyzed by means of immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis(2 D-PAGE).The 2-DE maps were visualized after coomassie blue staining and analyzed using PDQuest analysis software.Identification of differential protein spots was conducted by MALDI-TOF-MS,and the Mascot query software was used to search Swiss-Prot database for bioinformatics analysis.Western blotting was used to verify the expression of some differentially expressed proteins.Results:A total of 1 000 spots were identified in 2-DE maps of rat spinal cord tissues from the MT group and the NS group,and 36 proteins were significantly differentially expressed in the MT group compared with the NS group.Identification was conducted by MALDI-TOF-MS and Swiss-Prot database through Mascot query software,and a total of 14 proteins were obtained.Among them,2 protein spots were down-regulated in the MT group compared with that in the NS group,and 12 protein spots were up-regulated in the MT group compared with that in the NS group.Two kinds of proteins(NUDAA,ENOG) were verified by Western blotting and the results were consistent with proteomics data.Conclusion:The optimized 2-DE profiles for the proteome of spinal cord tissue in rats with chronic morphine tolerance is established preliminarily,which showed that morphine tolerance can cause changes in the expression of various proteins in the spinal cord.
Objective:To establish a two-dimensional gel electrophoresis(2-DE) map for comparative proteomic analysis of rat spinal cord with chronic morphine tolerance,and to detect differentially expression proteins that are associated with chronic morphine tolerance.Methods:Sixteen male SD rats received the intrathecal catheterization operation and they were randomly divided into a morphine tolerance group(MT group,n=8) and a saline group(NS group,n=8).The lumbar enlargement segments of the MT group and the NS group spinal cord were harvested and proteins were separated by 2-DE.Differential proteome profiles were established and analyzed by means of immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis(2 D-PAGE).The 2-DE maps were visualized after coomassie blue staining and analyzed using PDQuest analysis software.Identification of differential protein spots was conducted by MALDI-TOF-MS,and the Mascot query software was used to search Swiss-Prot database for bioinformatics analysis.Western blotting was used to verify the expression of some differentially expressed proteins.Results:A total of 1 000 spots were identified in 2-DE maps of rat spinal cord tissues from the MT group and the NS group,and 36 proteins were significantly differentially expressed in the MT group compared with the NS group.Identification was conducted by MALDI-TOF-MS and Swiss-Prot database through Mascot query software,and a total of 14 proteins were obtained.Among them,2 protein spots were down-regulated in the MT group compared with that in the NS group,and 12 protein spots were up-regulated in the MT group compared with that in the NS group.Two kinds of proteins(NUDAA,ENOG) were verified by Western blotting and the results were consistent with proteomics data.Conclusion:The optimized 2-DE profiles for the proteome of spinal cord tissue in rats with chronic morphine tolerance is established preliminarily,which showed that morphine tolerance can cause changes in the expression of various proteins in the spinal cord.
引文
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[18]Abdel-Zaher AO,Abdel-Rahman MS,Elwasei FM.Blockade of nitric oxide overproduction and oxidative stress by Nigella sativa oil attenuates morphine-induced tolerance and dependence in mice[J]Neurochem Res,2010,35(10):1557-1565.
[19]Muscoli C,Cuzzocrea S,Ndengele MM,et al.Therapeutic manipulation of peroxynitrite attenuates the development of opiateinduced antinociceptive tolerance in mice[J].J Clin Invest,2007117(11):3530-3539.
[20]Payabvash S,Beheshtian A,Salmasi AH,et al.Chronic morphine treatment induces oxidant and apoptotic damage in the mice liver[J]Life Sci,2006,79(10):972-980.
[21]Alves-Bezerra M,Majerowicz D,Grillo LA,et al.Serotonin regulates an acyl-CoA-binding protein(ACBP)gene expression in the midgut of Rhodnius prolixus[J].Insect Biochem Mol Biol,2010,40(2):119-125.
[22]Shibasaki M,Katsura M,Torigoe F,et al.Increase in diazepam binding inhibitor expression by sustained morphine exposure is mediated via mu-opioid receptors in primary cultures of mouse cerebral cortical neurons[J].J Neurosci Res,2007,85(13):2971-2980.
[23]Song Z,Guo Q,Zhang J,et al.Proteomic analysis of PKCgammarelated proteins in the spinal cord of morphine-tolerant rats[J].PloSOne,2012,7(7):e42068.
[2]Mercadante S.Problems of long-term spinal opioid treatment in advanced cancer patients[J].Pain,1999,79(1):1-13.
[3]Zou W,Xu W,Song Z,et al.Proteomic identification of an upregulated isoform of annexin A3 in the spinal cords of rats in a neuropathic pain model[J].Front Neurosci,2017,11:484.
[4]袁立霞,白殊同,李青原,等.Mtb诱导的佐剂性关节炎大鼠滑膜病变的蛋白质组学研究[J].南方医科大学学报,201434(9):1272-1276.YUAN Lixia,BAI Shutong,LI Qingyuan,et al.Proteomics study of synovial lesions in rats with Mtb-induced rheumatoid arthritis[J]Journal of Southern Medical University,2014,34(9):1272-1276.
[5]邹望远,李茂玉,郭曲练,等.神经病理性痛大鼠脊髓蛋白质组图谱的建立[J].中华麻醉学杂志,2008,28(12):1093-1096.ZOU Wangyuan,LI Maoyu,GUO Qulian,et al.Establishment of the proteomics of spinal cord in rats with neuropathic pain[J].Chinese Journal of Anesthesiology,2008,28(12):1093-1096.
[6]Charlton E.Ethical guidelines for pain research in humans Committee on ethical issues of the international association for the study of pain[J].Pain,1995,63(3):277-278.
[7]Poon YY,Chang AY,Ko SF,et al.An improved procedure for catheterization of the thoracic spinal subarachnoid space in the rat[J].Anesth Analg,2005,101(1):155-160.
[8]Wang HY,Friedman E,Olmstead MC,et al.Ultra-low-dose naloxone suppresses opioid tolerance,dependence and associated changes in mu opioid receptor-G protein coupling and gbetagamma signaling[J].Neuroscience,2005,135(1):247-261.
[9]Yoburn BC,Gomes BA,Rajashekara V,et al.Role of G(i)alpha2-protein in opioid tolerance and mu-opioid receptor downregulation in vivo[J].Synapse,2003,47(2):109-116.
[10]Rozenfeld R,Abul-Husn NS,Gomez I,et al.An emerging role for the delta opioid receptor in the regulation of mu opioid receptor function[J].Sci World J,2007,7:64-73.
[11]Raval AP,Dave KR,Mochly-Rosen D,et al.Epsilon PKC is required for the induction of tolerance by ischemic and NMDA-mediated preconditioning in the organotypic hippocampal slice[J].J Neurosci2003,23(2):384-391.
[12]Ferrini F,Trang T,Mattioli TA,et al.Morphine hyperalgesia gated through microglia-mediated disruption of neuronal Cl(-)homeostasis[J].Nat Neurosci,2013,16(2):183-192.
[13]Motin VG.Action of colchicine on analgetic effects of morphine and DADLE in rats[J].Biull Eksp Biol Med,1990,110(8):168-170.
[14]Borsodi A,Toth G.Microtubule disassembly increases the number of opioid receptor binding sites in rat cerebrum membranes[J]Neuropeptides,1986,8(1):51-54.
[15]Marie-Claire C,Courtin C,Roquse BP,et al.Cytoskeletal genes regulation by chronic morphine treatment in rat striatum[J]Neuropsychopharmacology,2004,29(12):2208-2215.
[16]Rex CS,Gavin CF,Rubio MD,et al.Myosin IIb regulates actin dynamics during synaptic plasticity and memory formation[J]Neuron,2010,67(4):603-617.
[17]Sekino Y,Kojima N,Shiraao T.Role of actin cytoskeleton in dendritic spine morphogenesis[J].Neurochem Int,2007,51(2/3/4):92-104.
[18]Abdel-Zaher AO,Abdel-Rahman MS,Elwasei FM.Blockade of nitric oxide overproduction and oxidative stress by Nigella sativa oil attenuates morphine-induced tolerance and dependence in mice[J]Neurochem Res,2010,35(10):1557-1565.
[19]Muscoli C,Cuzzocrea S,Ndengele MM,et al.Therapeutic manipulation of peroxynitrite attenuates the development of opiateinduced antinociceptive tolerance in mice[J].J Clin Invest,2007117(11):3530-3539.
[20]Payabvash S,Beheshtian A,Salmasi AH,et al.Chronic morphine treatment induces oxidant and apoptotic damage in the mice liver[J]Life Sci,2006,79(10):972-980.
[21]Alves-Bezerra M,Majerowicz D,Grillo LA,et al.Serotonin regulates an acyl-CoA-binding protein(ACBP)gene expression in the midgut of Rhodnius prolixus[J].Insect Biochem Mol Biol,2010,40(2):119-125.
[22]Shibasaki M,Katsura M,Torigoe F,et al.Increase in diazepam binding inhibitor expression by sustained morphine exposure is mediated via mu-opioid receptors in primary cultures of mouse cerebral cortical neurons[J].J Neurosci Res,2007,85(13):2971-2980.
[23]Song Z,Guo Q,Zhang J,et al.Proteomic analysis of PKCgammarelated proteins in the spinal cord of morphine-tolerant rats[J].PloSOne,2012,7(7):e42068.