重组人BDNF对Alzheimer病模型神经元作用的实验研究

BDNFAlzheimer,,,,(,710061):(rhBDNF)Alzheimer(AD)rhBDNF20molL-1A2535,(ACHE)MTT59.5%,(P0.01)rhBDNF65.7%72.6%,(P0.05),50ngmL-1rhBDNF,,Ca2+]irhBDNF,;AD,,A,AD:Alzheimer;;;:R749.1+6:A:167128259(2003)0120005205ExperimentalstudyontheeffectsofrecombinatehumanBDNFintheculturedneuronsofAlzheimersdiseasemodelLiuZhaohui,HuHaitao,MaDongliang,XuJiehua,YangPengbo(DepartmentofAnatomy,MedicalCollegeofXianJiaotongUniversity,Xian710061,China)ABSTRACT:ObjectiveToinvestigatetheeffectsofrecombinatehumanBDNF(rhBDNF)onnormalprimeculturedhippocampalneuronsandneuronsofAlzheimersdisease(AD)model.Methods20molL-1A2535toculturedhippocampalneuronsbeforeitwastreatedwithrhBDNF.Theresultswereobtainedbymorphologicalobservation,ACHEhistochemistry,confocalmicroscopyobservationandMTTmethods.ResultsTheneuronsurvivalrateincontrolgroupdecreasedto59.5%,whichshowedsignificantdifferencecomparedwiththatofblankgroup(P0.01).TheneuronsurvivalrateinrhBDNFexperimentalgrouproseto65.7%72.6%,andthereappearedasignificantdifferencebetweenexperimentalgroupandcontrolgroup(P0.05).TheoptimumconcentrationofrhBDNFwas50ngmL-1.ThequantityofcholinergicneuroninrhBDNFexperimentalgroupincreased,diameterofcellbodyandlengthofprocessesaugmented,andCa2+]icomparativelystable.ConclusionrhBDNFhasneurotrophiceffectsonnormalculturedhippocampalneuronsandcanprolongthelivingtimeofthecell.InculturedhippocampalneuronsofADmodel,rhBDNFhastheprotectivefunction,anditcandecreasecellmortalityrategreatly,weakenthetoxicitycausedbytheAandhelppreventandcureAD.KEYWORDS:Alzheimersdisease;2amyloidprotein;BDNF;hippocampalneuron:2002204222:2002210203:(19712),(),,.(Alzheimersdisease,AD),(SP)(NFT)(A)SP1,(APP),AD,AD2,NFTA,ALooA3;,AD,,A2535AD4,,(),NMDA24120032()JournalofXianJiaotongUniversity(MedSci)Vol.24No.1Feb.2003Ca2+,Ca2+,Ca2+]i,Ca2+]i,Ca2+]i,AD,Ca2+]iAD,,,,BDNFNT35,ADBDNFrhBDNFAD,rhBDNF11.1A2535(),DMEM(Hyclone),(GIBCO),(Sigma),()rhBDNF:GeneBank,DNAPCRBDNF(hBDNF)pBV220/hBDNF,pBV220/hBDNFDH5,hBDNF6A:A2535DMEM,1mmolL-1,0.2m3724h,A7,4,1.21.2.113dSD,75%()5min,,,;302.5gL-137,20min,15%(),,1000rmin-110min,;D2Hank2,15%(),,200,1106mL-11.2.2rhBDNF(MTT)1106mL-196,100L,CO224h,(10molL-1),,3d,rhBDNF,200100502512.5ngmL-1,DMEM,(rhBDNF),9d,MTT(5mgmL-1)20L,374h,,150L(DMSO),,490nm,(A)(),ArhBDNF,12d15dMTT1.2.3rhBDNFAD(MTT),,24h,3d,rhBDNF,1005025ngmL-1,DMEM,8d,A,20molL-124h,MTT1.2.4,9d40gL-120min,KarnovskyRoots(ACHE),(20)20ACHE,551.2.5Ca2+:Fluo23(AM)AM,,Ca2+,50100,Ca2+i8:,9d,Fluo23AM10gmL-1,,,3730min,23,1.2.61.2.21.2.31.2.4SPSS10.0,6()24,…xs;1.2.5Bio2QAnalyzeProgram22.14h,24h80%,,,12h,24h,,,,,,23d,,,78d,,,,,,(1),,1012d,,,,,,,20d,rhBDNF,,10d,,8d,A24h,,,,,,,(2);rhBDNF(3)2.2rhBDNF1rhBDNF9d12d15dBlank0.7870.0460.5320.0680.4260.056rhBDNF(200ngmL-1)0.7530.038--rhBDNF(100ngmL-1)0.7910.0480.7240.06130.6290.05533rhBDNF(50ngmL-1)0.8460.0590.7860.05230.7110.06433rhBDNF(25ngmL-1)0.8040.0550.7260.04330.6830.07533rhBDNF(12.5ngmL-1)0.7740.049--:3P0.05,33P0.019dMTT,,,,(1)2.3rhBDNFAD20molL-1A253524h,A100%,59.5%,(P0.01)rhBDNF,65.7%72.6%,6.2%13.1%,rhBDNF50gL-1,(P0.05,P0.01)(2)2rhBDNFADA/%Blank0.7820.054100Control0.4650.04959.54.1#rhBDNF(100gL-1)0.5140.05565.72.93rhBDNF(50gL-1)0.5680.05172.65.233rhBDNF(25gL-1)0.5500.03370.33.53:#P0.01;3P0.05,33P0.012.4rhBDNF68.75-1,58.37-1,(3)3(-1)/m/mBlank82.639.3715.682.93119.6810.69Control58.377.13#9.532.41#47.388.38#rhBDNF(50gL-1)68.757.37311.142.36391.578.743:#P0.01;3P0.052.5,,Ca2+]i,(4a),;(4b),Ca2+]i78,;(4c),Ca2+]i45,71,,,1BDNFAlzheimer18d(20)2A(40)3rhBDNFAAD(20)4abcrhBDNF3BDNF,,ADBDNF,BDNF,AD9,,rhBDNF,20molL-1A253524h,59.5%,,Ca2+]i,,AD,rhBDNF,rhBDNFAD,A,65.7%72.6%,6.2%13.1%,,,Ca2+]i,AAD,:NMDACa2+,Ca2+]i;,,,,Ca2+]i10;Ca2+]i::Ca2+,;Ca2+,:Ca2+,Ca2+,Ca2+,ATPpH,Ca2+]i,Ca2+]i,,Ca2+]iHarkany11A1-42Ca2+,BDNFTrk,,,,,BDNF,12(13)8()245pBV220/A2HBcAg1:200bpDNAMarker;2:DNA/HindMarker;3:EcoR+BamH;4:Nde;5:7Z/A2HBcAg:[1]SchenkD,BarbourR,DunnW,etal.Immunizationwithamyloid2betaattenuatesAlzheimer2disease2likepathologyinthePDAPPmouseJ.Nature,1999,400(6740):173-177.[2],1M.2,:,1992.19-59.[3MorganD,DiamondDM,GottschallPE,etal.ApeptidevaccinationpreventsmemorylossinananimalmodelofAlzheimersdiseaseJ.Nature,2000,498:982-985.[4BardF,CannonC,BarbourR,etal.Peripherallyadminis2teredantibodiesagainstamyloid2peptideenterthecentralnervoussystemandreducepathologyinamousemodelofAlzheimersdiseaseJ.NatureMedicine,2000,6(8):916-919.[5DeMattosRB,BalesKR,CumminsDJ,etal.Peripheralanti2AantibodyaltersCNSandplasmaAclearanceandde2creasesbrainAburdeninamousemodelofAlzheimersdis2easeJ.ProcNatlAcadSciUSA,2001,98(15):8850-8855.[6AlonM,RuthM,Victorzota,etal.Immunehyporespon2sivenesstoamyloid2peptideinamyloidprecursorproteintransgenicmice:implicationsforthepathogenesisandtreat2mentofAlzheimersdiseaseJ.ProcNatlAcadSciUSA,2001,98(18):10273-10278.[7LachmannS,MeiselH,MuselmannC,etal.Characteriza2tionofpotentialinsertionsitesinthecoreantigenofhepatitisBvirusbytheuseofashort2sizedmodelepitopeJ.Intervi2rology,1999,42(1):51-56.[8],1S2(HBVPreS2epitope)(HBcAg)J.,1994,10(3):221-227.[9],1M.:,1998.259.[10],,,1/RNARNAJ.,2002,18(1):29-33.()(8),,;,rhB