关于氨基酸和神经肽与疼痛关系的研究.ppt

,TatLeangLEEDepartmentofAnaesthesiaYongLooLinSchoolofMedicineNationalUniversityofSingapore,AdvancesinAminoAcidsandNeuropeptidesResearchandPain关于氨基酸和神经肽与疼痛关系的研究,Rationaltreatmentofpainrequiresidentificationofpainmechanismsastargetsofdrugtherapy,致病因素,临床实体,疾病机制,症状体征,疼痛机制,基于疼痛机制的治疗,与疼痛相关的症状体征,特定机制结果的评测,Rationaltreatmentofpainrequiresidentificationofpainmechanismsastargetsofdrugtherapy,致病因素,临床实体,疾病机制,症状体征,疼痛机制,与疼痛相关的症状体征,基于疼痛机制的治疗,特定机制结果的评测,Keymolecularelementsinthetransduction,transmissionandsignalprocessingofnociceptiveinputintheperipheralandcentralnervoussystemrepresentpotentialtargetsforthedevelopmentofnewanalgesics.,Mechanism-basedpaindiagnosis:issuesforanalgesicdrugdevelopmentWoolfCJ,MaxMB.Anesthesiology2019;95:241-9,Pain:movingfromsymptomcontroltowardmechanism-specificpharmacologicmanagementWoolfCJ.AnnInternMed.2019;140:441-5,Canweconquerpain?ScholJ,WoolfCJ.Natureneurosciencesuppl.2019;5:1062-7,基于病理机制的疼痛诊断:镇痛药发展的问题,我们能否征服疼痛?,疼痛:从症状控制向特异机制的药理学治疗转移,Background背景,Cerebralspinalfluid(CSF)reflectsmetabolicstateofbrain脑脊液反映疼痛的新陈代谢状态AnalysisofCSFaminoacidsandpeptideshighlyrelevantinpainperceptionandalsoneurologicaldiseases脑脊液中氨基酸和缩氨酸与疼痛感知和神经系统疾病高度相关ChronicPaininflammatory,neuropathyandcancer慢性疼痛分类发炎，神经系病，癌症Mechanismsverychallengingandnotyetunderstood对于疼痛机制，目前还没有被足够的了解，非常具有挑战性Lackofeffectivetherapeutics缺乏有效的治疗法,Background,Excitatoryandinhibitoryaminoacidsandalsonitricoxide(NO)areinvolvedinpainmodulation.兴奋性，抑制性氨基酸和一氧化氮涉及到对疼痛的调节Neuropeptidesareimplicatedinpainmodulation.神经肽影响对疼痛的调节,Objectives,AtwoprongedstudyonCSF对脑脊液的两次研究ToquantitativelyanalyzephysiologicalaminoacidsandpainrelatedcompoundsinCSFsamplesfrompainpatients对疼痛病人脑脊液中生理氨基酸和疼痛相关的化合物定量研究Tofindnewpain-relatedpeptides(molecularmass15,000Da)fromCSFsamplesofchronicpainpatients从慢性疼痛病人的脑脊液中寻找与疼痛相关的肽（分子量小于15000）Resultsshouldleadtobetterunderstandingofpainmechanismsandnewtreatmentstrategy实验结果帮助我们更深的了解疼痛机制和寻找新的治疗方法,AminoacidsinCSFandPain脑脊液中氨基酸和疼痛关系的研究,液相色谱法对生理氨基酸在未被预处理的脑脊液中的定量测量,TypicalChromatogram典型色谱层析图,STDAA氨基酸分离标准图,1-aspartate天(门)冬氨酸盐2-glutamate谷氨酸3-asparagine天冬酰胺酸4-glutamine谷酰胺5-citrulline瓜氨酸6-serine丝氨酸7-threonine苏氨酸8-glycine甘氨酸9-alanine丙胺酸10-arginine精氨酸11-taurine牛磺酸12-GABA伽马氨基丁酸13-proline脯氨酸14-valine缬氨酸15-methionine蛋氨酸16-isoleucine异亮氨酸17-leucine亮氨酸18-tryptophan色氨酸19-phenylalanine苯基丙氨酸20-cystine胱氨酸21-Ammonia氨22-lysine赖氨酸23-histidine组氨酸24-tyrosine酪氨酸,1-aspartate天(门)冬氨酸盐2-glutamate谷氨酸3-asparagine天冬酰胺酸4-glutamine谷酰胺5-citrulline瓜氨酸6-serine丝氨酸7-threonine苏氨酸8-glycine甘氨酸9-alanine丙胺酸10-arginine精氨酸11-taurine牛磺酸12-GABA伽马氨基丁酸13-proline脯氨酸14-valine缬氨酸15-methionine蛋氨酸16-isoleucine异亮氨酸17-leucine亮氨酸18-tryptophan色氨酸19-phenylalanine苯基丙氨酸20-cystine胱氨酸21-Ammonia氨22-lysine赖氨酸23-histidine组氨酸24-tyrosine酪氨酸,CSFsample,Salientfeaturesofthemethod方法特色,UseofTritonX-100（TritonX-100的应用）Nopre-treatmentofCSF脑脊液不被预处理Enhancedrecoveryofhydrophilicaminoacids提高亲水性氨基酸的恢复Detectionofcitrullineimproved提高对瓜氨酸的检测RequiressmallvolumeofCSF-10-20L使用较少量的脑脊液20LofCSFsampleyields140Lofdabsylatedsample.20微升脑脊液样本经过dabsylation产生140微升样本Injectionvolume20L一次样本液相色谱分析只需20微升6repeatedanalysisofsamesampleoncedabsylatedispossible能够对同一Dabsylate过的样本进行6次重复分析Qeffectivelyseparated-100timeshigherconcentrationinCSF谷酰胺被有效分离在脑脊液中浓度是其它氨基酸的100倍StabilityofdabsylderivatesDabsyl衍生物稳定性Dabsylatedsamplestableforoneweekin4Candfor48hrsatroomtemperature经过Dabsylation的样本能够在4度保持一周，室温下保持48小时,Analysisofaminoacidsandnitricoxideinlaborpain对产前疼痛病人脑脊液中氨基酸和一氧化氮的分析,Differentialaminoacidprofileinthecerebrospinalfluidofparturientswithandwithoutlaborpain对产前疼痛和非疼痛产妇脑脊液中氨基酸量不同的分析LeeTL,SethuramanR,ChuiJW,TachibanaSAnnualmeetingoftheAmericanSocietyofAnesthesiologists,23-27Oct2019,LasVegas,USA,ConcentrationsofpainrelatedaminoacidsinCSFofpregnantwomen怀孕妇女脑脊液中和疼痛相关的氨基酸浓度,ValuesareexpressedasmeanSEM;aPregnantwomenwithnolaborpain;bPregnantwomeninacutelaborpain(P0.05)*(P0.01)*(P0.001)Statisticallysignificantwhencomparedtothecontrolgroup.产前急性疼痛b与产前无疼痛a相比具有统计价值,StatisticalcorrelationbetweenPainintensity(PI)andtheconcentrationofpainrelatedaminoacidsincerebrospinalfluidofthelaborpaingroupascomparedagainstthenopaincontrol.疼痛强度和与疼痛相关氨基酸在产妇脑脊液中浓度的统计相关性,PIPainintensitygradedasMild轻度(n=11),Moderate中度(n=18),Severe严重(n=9)*(P0.05)*(P0.01)*(P0.001)Statisticallysignificantcorrelationrelativetothecontrolgroup.,CSFconcentrationofotheraminoacidsnotrelatedtopain与疼痛不相关的氨基酸在脑脊液中浓度,ValuesareexpressedasmeanSEMaPregnantwomenwithnolaborpainbPregnantwomeninacutelaborpain*(P0.05)*(P0.01)*(P0.001)Statisticallysignificantwhencomparedtothecontrolgroup.,Results结果,Aspartateisinvolvedinlaborpainbutnotglutamate天冬氨酸盐与产前疼痛相关，谷氨酸不相关Inhibitoryaminoacidsglycine,GABA,taurineinvolvedinlaborpain抑制性氨基酸如甘氨酸，伽马氨基丁酸，牛黄酸与产前疼痛相关NOmightbeinvolvedinlaborpainmayhavedifferentialroles一氧化氮可能涉及至产前疼痛，但可能有不同作用,Acomparativestudyontherolesofaminoacidsandnitricoxideinchronicandacutepain氨基酸和一氧化氮在慢性和急性疼痛中的作用对比研究,Relationshipbetweenphysiologicalaminoacidsincerebrospinalfluidandpain.脑脊液中生理氨基酸和疼痛的联系TachibanaS,SethuramanRandLeeTLIn:AnnaCapasso(Ed)RecentDevelopmentsinPainResearch,ResearchSignpost;2019,ppISBN81-308-0012-8.,Concentrationofpain-relatedaminoacidsinCSFChronicpainVsControlgroupandChronicpainVsAcutepaingroup和疼痛相关的氨基酸在脑脊液中浓度慢性疼痛组和标准组，慢性疼痛组和急性疼痛组,ValuesareexpressedasmeanSEM;*,(P0.05)*,(P0.01)*,(P0.001)Statisticallysignificantwhencomparedtothechronicpaingroup与慢性疼痛组相比有统计.Citrullinedetectedinonly3outof35controlCSF,17outof29chronicpainCSFand22outof46acutepainCSF.在标准组（35样本）中，仅有3个样本中含瓜氨酸；29个慢性疼痛病人中17个含有瓜氨酸；46个急性疼痛病人中有22个含瓜氨酸,ComparisonbetweenPainrelatedaminoacidsinCSFofChronicandAcutepainpatients-DifferentialrolesfortheseaminoacidsinthetwotypesofPainstates和疼痛相关的氨基酸在慢性和急性病人脑脊液浓度对比这些氨基酸在两种不同疼痛状态中的作用*(P0.05)*(P0.01)*(P0.001)StatisticallysignificantdifferencesbetweenthetwogroupscomparedGlutamine(Gln)concentrationsareexpressedasx10-2mol/L,Results,ChronicpainVsNopaingroup慢性疼痛和无疼痛Aspartate,glutamate,glycineandNOinvolvedinchronicpainperception天门冬氨酸盐，谷氨酸，甘氨酸和一氧化氮与疼痛的感知有关ChronicpainVsAcutepainDifferentialrolesofaminoacidsinchronicandacutepainstates慢性疼痛和急性疼痛氨基酸在慢性和急性疼痛状态的不同作用Aspartate-akeyplayerinpathogenesisofchronicpain天门冬氨酸盐导致慢性疼痛的重要因素,FutureScope前景,ThissimpleCSFaminoacidanalysismethodcanbeappliedtostudyvariousotherpainstatesandCNSdiseases.这种简易的氨基酸分析方法可以用于其它疼痛状态和神经系疾病分析ThismethodofCSFanalysiswithoutpretreatmentinthepresenceofTritoncanalsobeappliedtootheranalysisprocedures.这种含有Triton，无预处理脑脊液分析方法可以用于其它分析方法中Withmorenumberofagematchedsubjects(increase“n”)andwithwelldefinedpainconditionspotentialmarkerscanbeidentifiedforeachtypeofpain.同样年龄组中如果有足够样本，在有明确的对疼痛定义的前提下，对各种疼痛类型，可以鉴别出潜在的生物标记,PurificationandcharacterizationofpeptidesfromCSFofchronicpainpatients慢性疼痛病人脑脊液中肽的提纯和特性研究,PeptidePurificationStrategy肽的提纯方法,Identification鉴别,Characterisation特性,Quantification量化,Localisation位置,CNSeffects在中枢神经中的影响,Antagonists对抗剂,Painmodels疼痛原型,TheStory,1973morphinebindingsites/opiatereceptorsLTerenius,SSnyder,ESimon,1975enkephalinJHughes,HKosterlitz,Papaversomniferumpain,diarrhoea377:532-35,Reinscheidetal.Science2019;270:792-94,NOCICEPTIN(Noc),ORPHANINFQ(OFQ),阿片孤儿受体（ORL-1）的内源性配体,孤啡肽,孤啡肽,RatbrainnociceptinMice(i.c.v.)hyperalgesia,PorcinebrainOrphaninFQMice(i.c.v.)hyperalgesia(tail-flickassay)decreaselocomotoractivity,大鼠脑,猪脑,痛觉过敏(甩尾试验)减少运动的活动性,Noc/OFQ,HeptadecapeptideStructurallysimilartodynorphinAi.t.ori.c.v.administration:-hyperalgesia(hotplate100m(c,d).,Okuda-AshitakaEetal.2000,Spinaltrigeminaltract,paramedianraphenucleus,Ventromedialnucleusofhypothalamus,NSTreceptor(?),Doesnotactontheclassicopiatereceptors,DoesnotactonORL1receptor,痛稳素受体(?),不作用于典型的阿片受体,不作用于阿片孤儿受体(ORL1)受体,TheroleoftheORL1receptorinthemodulationofspinalNeurotransmissionbynociceptin/orphaninFQandnocistatinAhmadiS,LiebelJT,ZeilhoferHU.Eur.J.Pharmacl.2019;412:39-44,Modulationofsynaptictransmissionbynociceptin/orphaninFQandnocistatininthespinalcorddorsalhornofmutantmicelackingthenociceptin/orphaninFQreceptorAhmadiS,KotallaC,GuhringH,TakeshimaH,PahlA,ZeilhoferHUMolecularPharmacology2019;59:612-8,PossiblepathwaysinvolvedinNoc/OFQ-inducedhyperalgesiaandallodyniainthespinalcord,ItoSetal.2000,ClinicalSignificance,Anti-nociceptiveResponsesProducedbyHumanPutativeCounterpartofNocistatin,MinamiTetalBrJPharmacol2019;124:1016-18,IdentificationofNocistatininHuman,RatandMouseBrainandHumanCerebrospinalFluid,LeeTLetal.NeuroReport2019;10:1537,临床意义,对抗由人们公认的痛稳素类似体所引起的伤害性反应,人,大鼠,小鼠大脑及人脑脊液中痛稳素的鉴别,Identi