【doc】 碱性成纤维细胞生长因子对大鼠局灶性脑缺血再灌注后脑细胞内游离钙变化的影响

【doc】 碱性成纤维细胞生长因子对大鼠局灶性脑缺血再灌注后脑细胞内游离钙变化的影响 碱性成纤维细胞生长因子对大鼠局灶性脑 缺血再灌注后脑细胞内游离钙变化的影响 192 E仃ectofbasicfibroblast freecalciumfollowingfocal BaiHong—ying,WenGong—ling,LouJi—yu 中国临床康复第9卷第29崩2005—08—07出版 ChineseJournalofClinicalRehabilitation,August72005Vo1.9No29 ? BAsICREsEARCH? growthfactoronratbrainintracellular ischemia-reperfusioninjury DepartmentofNeurology.SecondAmliatedHospitalofZhengzhou University,Zhengzhou450014,HenanPmvim,e,China Correspondenceto:BaiHong—ying,Chiefphysician,Departmentof Neurology,SecondAffiliatedHospitalofZhengzhouUniversity,Zhengzho u 450014.HenanPrayinee.Chinahvbai@126.cam Received:2004—12—17Accepted:2005一O6一l0f15/SM1 Abstract BACKGROUSD:BasicfibroblastgrowthfactorfbFGnpossesses multiplefunctionssuchaspromotingneuronalsurvivalandgrowthofeell processesinvitroandantagonizingthetoxicityofexcitatoryaminoacids, therebyplayingimportrolesinfunctionalrecoveryofthecentralnervous systemfCNS).ButwhetherbFGFoffersneuroprotectiononisehemiebrain tissuesbymodulatingintracellularfreecalciumcontentremaimsunknown. OBJECTIVE:FoexploretheeffectofbFGFonintracellularfreeCain theneuralcellsintheeventoffocalcerebralischemia—reperfusionfIR1 1i1jur DESIGN:Randomizedcontrolledstudv. SETr?,iG:I)epartmentofNeurologyofSecondHospitalAffiliatedto ZhengzhouUniversity. MATERIALS:ThisstudywasconductedjntheLaboratoryofthe DepartmentofNeurology,SecondHospitalAffiliatedtoZhengzhou UniversitybetweenAugustandDecember2003.Totally24SDwere randomizedintoshamoperationgroup,ischemicgroup,1RgroupandbFGF exposuregroupwithrats1neachgroup. M|ETH0DS:MiddlecerebralarteryocclusionfMCAO1modelwas establishedinratsinIRgroupandbFGFexposuregroupbyinducing artefia1thrombosiswiththread.whichwasnotpreformedinratsi13the shamoperationgroup.RatsinbFGFexposuregroupreceived intraperitonealinjectionof10g/kgbFGFimmediatelyafterischemia, whichwasreplacedbythesamevolumeofphysicalsalimeintheothertwo groups.FreeCainbraincellswasdetectedat24hoursof1R. MAIN0UTC0MEMEASURES:FreeCa:inthebraincellsat24 hoursof1R. RESULTS:AIlthe24ratssurvivedtheexperiment.FreeCainlR groupwassignificantlyhigherthanthatoftheshamoperationgroup [(673.46+18.44)us(224.71?1O58)nmol/I,,=1329.06,P<O.叭],and alsosignificantlyhigherinbFGFexposuregroup【(378.37+21.08)nmol/L, l329.06.P(0.0l1. C0NCLUS10N:Intracellularfreecalciumcanbeobviouslydepressedby bFGFfollowingIRinjury.whichbenefitscel1membranestabilityandhelp preventintracellularCa”overload. BoiHY,WenGL.LouJYEffeclofbasicfibroblaslgrowthfadoronralbrain inlracellularfreecalciumfollowingfocalischemia—reperfusioninjuryZho ngguo LinchuangKangfu2005;9(29):192——3(China)(wwwzglckfcom] INTRoDUCTIoN Basicfibroblastgrowthfactor(bFGF)isananionpolypeptide possessingmultiplebioactivitiessuchaspromotingneuronal survivalandgrowthofcellprocessesinvitroandantagonizingthe toxicityofexcitatoryaminoacids,therebyplayingimportantroles inthefunctionalrecoveryofthecentralnervoussystem(CNS).In recentyears,theroleofCa”duringcerebralischemia—reperfusion (IR)injuryhasreceivedincreasingattention,andCaisbelieved toparticipateinthepathologicalprocessofcerebralIRinjury….In thisstudy,,thread—inducedthrombosiswasadoptedtoestablish focalcerebralIRinjurymodelinrats,soastoexploreatthe cellularlevelthemechanismofbFGFagainstIRinjuryby detectingtheintracellularfleeCaconcentrationinthebrainof theratmodels MATERIALSANDMETHoDS Materials ThisstudywasconductedintheLaboratoryoftheDepartmentof Neurology,SecondHospitalAffiliatedtoZhengzhouUniversity betweenAugustandDecember2003.Totally24specific pathogen—fleehealthyadultSD.withbodymassof270—320g fmaleandfemalebyhalnwereprovidedbyHenanProvincial ExperimentalAnimalCenterfanimalcertificationnumber: 4l0l171andallowedfreeaccesstowaterandfood.Therats wererandomizedintoshamoperationgroup,ischemicgroup,IR groupandbFGFexposuregroupwith8ratsineachgroup. Establishmentofmiddlecerebralarteryocclusion(MCAO) model Theratswereanesthetizedwithl00g/Lhydrochlorideat300 mg/kg,andMCAOmodelwasestablishedbymeansofartery thrombosisindueedbythreadasdescribedbyLongaI:I.withthelefl middlecerebralarteryoccludedforlhourfollowedbyreperfusion for24hours.Themodelwasconsideredsuccessfulwhenclockwise circlingwhenwalkingandrightforelimbflexionuponholdingup bythetailoccurred.Theratsintheshamoperationgroupreceived allthesametreatmentbutMCAO. Medication bFGFwasproducedbyBeijingShuangluPharmacologicalCo.Ltd. TheratsinbFGFexposuregroupreceivedintraperitonealinjection ofdilutedbFGF(10kg)immediatelyafterischemia,whichwas replacedbythesamevolumeofphysicalsalineintheothertwo groups.FreeCainthebraincellswasdetectedat24hoursofIR. DetrminationofintracellularfCa”1iconcentration Preparationofratbraincellsuspension:Afterischemiaforlhour andreperfusionfor24hours.thelefthemisphereoftheratbrain wasquicklyremovedandimmersedincoldD—Hankssolution. Afterremovalofthesoftmeningesandvessels.thebraintissues wasrinsedwithpreeooledHankssolutionfor3—4times.digested withl-25g/Ltrypsinat37.Cfor30minutes.whichwas terminatedbyDMEMthatcontainingl5%caIfserum.filtered through200meshnylonsieveandcentrifugedatl000r/minute for5minutes.ThepelletswerethenrinsedwithcoldHanks solutiontwicebeforesuspendedwithDMEMcontainingl5%calf serumat2×l0/mLTnTpanbluestainingdemonstrateaviable cellratioof95%. AssavofthebraincellsusDensionf0rFura一2/AMloadand nuorescence:TwomillilitersofthecellsusDensionwasbathedat 37?f0r5minutes.vIbratedf0r45minutes37.Cer additionofFura一2/AMfatthenal(?oncentrationof5mol/L). ThecellsuspensionwithFura一2/AMloadwascentriflJgedat l(x)0r/minute, sedwithHankssolutioncontaining2g/L FBStwice.andthecellconcentrati0nwasadltedto2×l0/mL. Hitachi一85血再灌注后脑细胞内 “?w.kkf.coltl3385083@sinacom’’游离钙变化的影响 maximumfluorescencevalue.andthenEDTAwasaddedfor detectingFfinallyintracellularCawascalculatedaccording totheformula【Ca】,=Kd(F-F)/(F一一F)(Kd=224nmol/L). Statisticalanalysis:AIldatawereexpressedasMcan?SDand analyzedbythesecondwriterwithSPSS10.0statisticalsoftware. analysisofvariancewasusedforcomparison. RESULTS Quantitativeanalysisoftheexperimentalanimals Allthe24ratssurvivedtheexperimentandenteredthefinal analysiswithoutloss. Changesoffree【Ca”liconcentrationinthecorticalneuronsof ratbrain FreeCaconcentrationinIRgroupwassignificantlyhigher thanthatintheshamoperationgroup【(673.46?18.44)” (224.71?10.58)nmol/L,F=1329.06,P<0.011,also significantlyhigherthanthatinbFGFexposuregroup 『(378.37~21.08)nmo1/L,F--1329.06,P<0.01】. DISCUSSION LargeamountofCaaccumulatesintheneuralcellsafterIR injurytoresultinseveretoxicityandaseriesofpathological changes,promotingandenhancingsecondarybraininjury,whichis believedtobethefinalcommonpathwayofneuronaldeath[I.The resultsofthepresentstudyshowedthatfreeCainthebraincells obviouslyincreasedat24hoursofIR.Duringbrainischemia. oxidativephosphorylationintheneuralcellswasreduced. accompaniedbydecreasedsynthesisant1increasedassumptionof ATP.meanwhileNa一Kpumpfunctionwascompromisedas shownbydecreasedcellmembranerestingpotentialtoactivate voltage—dependentCa”channe1.whichresultsinmassiveCa influxthatcannotbepumpedoutduetoloweredpumpactivity. Ontheotherhand,morepre—membraneexcitatoryaminoacidwas releasedandactonN—methyl—D—asparticacidreceptor.also resultinginmassiveCainfluxduetotheopeningofreceptor— dependantCachannelI4j.Duringreperfusion.agreatdealoffree oxygenradicalswereproducedandlcadtomembrane lip0per0xidation,affectingthepermeabilityandiontransportation andfurtherenhancingworsenCa”overload. ExperimentalevidencesuggeststhatbFGFplaysaprotectiverole againstcerebralIRinjuryasaneurotrophicfactor.Inthisstudy weobservedthatbFGFcouldolwiouslyreducepost—IR intracellularfreeCa2leve1.similartotheobservationsin previousreportsimplyingtheprotectiveroleofbFGFin cerebralIRinjuryButthemechanismwasstillunclear.butit wassuggestedthatbFGFcouldmarkedlypromotetheexcitatory toxicityofglutaminenvitroculturedneural(:ells161.Western blottingandimmunocytochemistryprovethatbFGFcanattenuate theexpressionofN—methyl—D—asparticacidreceptor(M71000) inhippocampalneurons7I.whichmightbeoneofthe mechanismsbywhichbFGFattenuatesintracellularCa overload.Inaddition,bFGFcanincreasethecatalaseactivityof ischemicbraintissue18[,therebyeffectivelvreducingexcessive freeoxygenradicalsduringIR,inhibitinglipoperoxidation, 193 stablingcellmembraneandpreventingintracellularCa overload. REFERENCES BicklerPE.HansenBM.Causesofcalciumaccumulationinratcorticalbrain slicesdufin~hypoxiaandischemia:roleofionchannelsandmembrane damage.BranResI994;665(2):269—76 LongaEZ,WeinsteinPR,CarlsonS,etolReversiblemiddleeerebralartery occlusionwithoutcraniectomyinrats.StrokeI989;20fI1:84—91 MorleyP,HoganMJ,HakimAM.Calcium.mediatedmechanismsofischemi c injuryandprotection.Bra/nPathol1994;4(I1:37—47 CazalM.PietfiS.CulcasiM.eto1.NMDA.dependentsuperoxide Lafon— productionandneurotoxicity.NatureI993;364(6437):535—7 MattsonMP,ChengBGrowthfactorsprotectneuronsagainstexcitotoxic/ 1schemicdamagebystabilizingealeiumhomeostasis.StrokeI993;24fI2Supp1): I136—40 ZhangZJ,WangWD,WanO,eto1.TheeffecttobFGFonneurogenesisin brainfollowingcerebralisehemiainrats.ZhongguoLinchuangKartgfu(中 国临 床康复)2002;6(4):530一I MattsonMP,KumarKN,WangH,eto1.BasicFGFregulatestheexpressionofa functional7IkDaNMDAreceptorproteinthatmediatescalciumimquxand neurotoxicitvinhippocampalneurons.JNeuroscI993;13rII1:4575—88 IAuX,ZhuXZ,jiXQ.Effectofbasicfibrob|astgrowthfactorollfocalisehemic iniuryandantioxidantenzymeactivities.ZhongKu~YaoLBaoI999;20(3): 22—73I 碱性成纤维细胞生长因子对大鼠局灶性脑缺血 再灌注后脑细胞内游离钙变化的影响 白宏英,闻公灵,娄季宇(郑州大学第二附属医院神经内科,河南省郑 州市 450014) 通讯作者:白宏英,女,1964年生,河南省民权县人,汉族,1985年河南 医科大学毕业,学士,主任医师,主要从事脑血管疾病研究. 摘要 背景:碱性成纤维细胞生长因子可促进体外培养的神经元存活及突 起生长,拮抗兴奋性氨基酸毒性,对中枢神经系统功能恢复起重要作 用,能否通过影响脑细胞内游离钙离子浓度对缺血脑组织起保护作 用. 目的:从细胞水平探讨碱性成纤维细胞生长因子对大鼠局灶性脑缺血 再灌注损伤时神经细胞内游离Ca浓度变化的影响. :完全随机对照实验. 单位:郑州大学第二附属医院神经内科. 材料:实验于20o3—08/12在郑州大学第二附属医院神经内科实验室完 成.24只SD大鼠随机分为假手术组,缺血再灌注组和碱性成纤维细胞 生长因子治疗组,每组8只. 方法:缺血再灌注组及碱性成纤维细胞生长因子治疗组采用线栓法建 立大脑中动脉闭塞模型;假手术组除不插线外,余同其他两组.碱性成 纤维细胞生长因子治疗组于缺血后即刻腹腔注射1Og/kg碱性成纤维 细胞生长因子,其余两组腹腔注射等量生理盐水.各组大鼠于缺血再灌 注24h检测脑细胞游离钙浓度. 主要观察指标:各组大鼠缺血再灌注24h脑细胞游离钙浓度. 结果:24只大鼠全部进入结果.缺血再灌注组明显高于假手术组 【(67346~1844),(22471~10.58)nmol/L,(F=I32906,P<0O1)1.碱性 成纤维细胞生长因子治疗组明显低于缺血再灌注组『(37837~2108). (67346~1844)nmol/L(1329.06.P<0.01)1. 结论:碱性成纤维细胞生长因子能明显抑制大鼠缺血再灌注后脑组织 内游离钙水平,起到稳定细胞膜,防l细胞内钙超载的作用. 主题词:脑缺血;再灌注损伤;成纤维细胞生长因子2;钙 中图分类号:R743文献标识码:A文章编号:167I一5962一(2005)29—0I92—02 白宏英闻公灵娄季宇碱性成纤维细胞生长因子对大鼠局灶性脑缺血再灌注 后脑细胞内游离钙变化的影响【J】中国j缶床康复.2005,9(29):192—3 [wwwzglckfcoral (EditedbySongJW/SunSG/JiH/WangL) ?2345678