胃电刺激对ICC表型及ICC-ENS突触连接可塑性的影响

华中科技大学博士学位论文胃电刺激对ICC表型及ICC-ENS突触连接可塑性的影响姓名：李翠萍申请学位级别：博士专业：内科学（消化系病）指导教师：侯晓华2008040120084291interstitialcellofCajalICCentericNeverSystemENSgastricelectricalstimulationGES[1]ICCc-kitstemcellfactorSCF,SCF/KITICCICCICC[2]ICC[3]c-kitanti-c-kitantibodyACK2SCF/KITICCICC[4]ICC30~60ICCICC[5]ICC12ICC[6]SCF/KITP38[7]P38[8-9][10]3ICCICC-ENSICCICCICC60mg/kgn=36246810126(n=18)3ICC14c-kitdesminc-kitSMHCICCc-kit-SMAICCICC24ICCICC34c-kitmRNA2wkvs6wkvs12wk0.60±0.15vs0.39±0.15vs0.39±0.11,P0.05-SMAmRNA2wkvs6wkvs12wk1.19±0.08vs1.21±0.11vs1.19±0.13,P0.05desmin2wkvs6wkvs12wk0.58±0.11vs0.67±0.13vs0.78±0.14P0.05SMHC2wkvs6wkvs12wk0.34±0.08vs0.46±0.08vs0.63±0.11P0.05SMLC2wkvs6wkvs12wk0.24±0.07vs0.29±0.08vs0.31±0.06P0.05mRNAc-kit2wkvs6wkvs12wk:0.61±0.15vs0.61±0.12vs0.58±0.15,P0.054-SMA2wkvs6wkvs12wk1.25±0.09vs1.22±0.14vs1.22±0.13,P0.05desmin2wkvs6wkvs12wk0.58±0.11vs0.57±0.12vs0.51±0.13P0.05SMHC2wkvs6wkvs12wk0.31±0.06vs0.29±0.10vs0.31±0.06P0.05SMLC2wkvs6wkvs12wk0.23±0.06vs0.25±0.06vs0.21±0.08P0.05mRNA5c-kit2wkvs6wkvs12wk0.37±0.09vs0.24±0.08vs0.11±0.04P0.05-SMA2wkvs6wkvs12wk1.49±0.19vs1.43±0.24vs1.51±0.20P0.05desmin2wkvs6wkvs12wk0.72±0.10vs0.76±0.14vs0.84±0.15P0.05SMHC2wkvs6wkvs12wk0.53±0.14vs0.62±0.14vs0.65±0.16P0.05SMLC2wkvs6wkvs12wk0.11±0.02vs0.18±0.06vs0.29±0.06P0.05c-kit2wkvs6wkvs12wk0.44±0.10vs0.42±0.10vs0.37±0.0P0.05-SMA2wkvs6wkvs12wk1.50±0.22vs1.49±0.23vs1.52±0.22P0.05desmin2wkvs6wkvs12wk0.69±0.13vs0.67±0.14vs0.67±0.14P0.05SMHC2wkvs6wkvs12wk0.69±0.15vs0.69±0.08vs0.64±0.10P0.05SMLC2wkvs6wkvs12wk0.10±0.03vs0.09±0.03vs0.09±0.03P0.05ICCCajalChangesofphenotypeofICCindiabeticratsinducedbySTZAbstractAims:TheaimsofthisstudyweretoobservechangesofphenotypeofICCindiabeticratsinducedbySTZ.Methods:36diabeticratsand18controlswereusedinthisstudy,whichweresacrificedatthetimepoint2,4,6,8,10and12weeksafterinjectionofSTZorcitricacidbuffer.ThemorphologyofICCwasobservedbyelectronmicroscopyanddoublestainingof5immunohistochemistrywithantibodiesforc-kitandmuscle-specificfilamentproteins;OccurrenceofapoptosiswasalsoassayedbyTUNEL;RT-PCRandWesternblotwereusedtoconfirmtheexpressionsofmRNAandproteinforc-kitandmuscle-specificfilamentproteins.Results:1Kitanddesminorsmoothmusclemyosinareisolatedincontrolsbutcolocalizedindiabeticrats.Kitand-SMAimmunoreactivitieswerenotcolocalizedinbothcontrolsanddiabeticrats.2RemainingKit-immunopositivecellsinmyentericregiondevelopedultrastructuralfeaturessimilartosmoothmusclecells,includingprominentfilamentbundlesandexpressionofthemuscle-specificintermediatefilamentproteindesmin,andsmoothmusclemyosin.3Apoptosiswasnotdetectedinmyentericregion.4DecreasinginmRNAandproteinexpressionsofc-kitandincreasinginmRNAandproteinexpressionsofdesminandsmoothmusclemyosinheavyandlightchainweredetected.Conclusions:ThesedatasuggestedinherentplasticitybetweentheICCandsmoothmusclecellsindiabeticratsinducedbySTZ.KeyWords:phenotype;InterstitialcellsofCajal;smoothmuscle;Diabetesmellitus6[3]SD54,250-350g,n=36n=18STZ,Alexis,USA60mg/kg,1.5ml119.6mM/L24681012633mlELISART-PCRWesternBlot40.1M-,750W3minPBS5minX330minc-KitC-191:100sc-168SantaCruzUSAa-SMA1:100,Desmin1:100,21:400Abcam,USA4°C37°C30minPBS5minX3Cy31:100SigmaUSAFITC1:100KPL7UK37°C60minOlympusFV500JapanTUNEL40.1M-,750W1min20-25°CddH2O,20-25°CPBS3BSA200.1MpH7.5Tris-Hcl30minPBS5minX250µlTUNELRocheGermany50µl37°C60minOlympusFV500Japan12.55-1012520.1MPH7.4220130-1200.1M5070909090100251122801010TecnaiG212FEI,NetherlandsRTPCR100mg500µlTriZol™Invitrogen,USA4°C12,000g10min200µl10min12,000g10min200µl20°C30min12,000g10min75%200µl7,500g10minRNARTEPDEPC11µl,DEPCRNA12µl50µg/mlOligo(dT)2µlPCR(PTC-150MiniCycler™MJResearchUSA)870°C5min20U/µlRNsin1µl10mmol/LdNTP4µl5X8µl200U/ml2µlPCR37°C60min95°C5mincDNA20°CPCRPCR25µl2xTaqPCRMasterMix12.5µl2µlcDNA50pmol/LInvitrogen,USA0.3µl9.9µlddH2OPCR94°C5min94°C30s30s72°C30s3572°C7min14µlPCR1.2%100V15minImageJODOD/ß-actinODmRNAWesternBlot100mg490µl5µlPMSF5µlAprotin4°C12,000g10min-70°C10SDS-PAGE40µg/100V3.5h200mA5090minPVDFMILLIPOREIrelandPVDF0.1%Tween20TBS51hc-Kit(C-19)(1:200sc-168SantaCruz,USA)a-SMA(1:200)Desmin(1:200)2(1:400AbcamUSA)2(1:1000CellSignaling,USA)4°CActin(I-19)(1:200sc-1616-RSantaCruz,USA)0.1%Tween20TBS10minX31h10minX3ECLPIERCE,USA)SSPS,x±s,ANOVAP0.059ICCc-kit(1AGM)-SMA(1B)desmin(1H)SMHC(1N)c-kit-SMA(1C)c-kitdesmin(1I)c-kitSMHC(1O)c-kit(1DJP)-SMA(1E)desmin(1K)SMHC(1Q)c-kitdesmin(1L)c-kitSMHC(1R)(),c-kit-SMA(1F)ICC2ICCICC4ICCICCICCICC3Table1PrimersUsedintheDetectionofthemRNAsProductSize(Bp)Tm(°C)Primerß-actin189485'-AGGGAAATCGTGCGTGAC-3'5'-ACCCAGGAAGGAAGGCT-3'c-Kit500545'-CAAGAAGAACAGGCAGACGC-3'5'-TCCCAGAGGAAAATCCCATA-3'desmin334505'-GTCCTACACCTGCGAGATTGA-3'5'-GACTTCGGAACCCCTTTGCTC-3'a-SMA493545'-TTCAAGAAGGAGCGAAACAC-3'5'-GCCGCTCAATCAGTTCAATA-3'SMHC425525'-AAGGAAACACCAAGGTCAAG-3'5'-GAACGAATGGAGACAAATGC-3'SMLC537575'-GCTGGGCATTTTAGTATCT-3'5'-TTGACCCTATCTGAACCTC-3'10Figure1.ConfocalimagesofKit+cellsdouble-labeledwithantifilamentproteinsat4wkofdiabeticratandcontrols.ColocalizationofKitand-SMAimmunoreactivitiesinstomachofcontrol(A–C)anddiabeticrat(D–