简介：BACKGROUND:Withdevelopmentsoftissueengineeringandgeneticengineering,weaimtoculturemyoblasts,whicharecharacterizedbyhighpurity,highqualityandhighproduction,forwideapplicationinneuralregenerationresearches.OBJECTIVE:Tomodifytraditionaldissociationmethodinordertoobtainmyoblasts,whicharecharacterizedbyhighpurity,highqualityandhighproduction,andexplorethebiologicalpropertiesunderinvitroculture.DESIGN:Observationalstudy.SETTING:BasicInstituteofAcademyofMilitaryMedicalSciencesofChinesePLA.MATERIALS:FourneonatalWistarratsof5daysold,bothgendersandmeanbodymassof10gwereselectedinthisstudy.Themainreagentsanddevicesweredetailedasfollows:DMEMmedium(GibcoCompany),fetusbovineserum(FBS,HycolneCompany),collagenaseⅡ(SigmaCompany),trypsin(SigmaCompany),dispaseⅡ(SigmaCompany),desminantibody(FuzhouMaixinCompany),antibodyⅡandABCkit(WuhanBosterBiotechnologyCompany),deskcentrifuge(KUBATO,Japan),andinvertedphasecontrastmicroscope(LEICADMIRB,Germany).METHODS:TheexperimentwascarriedoutintheBasicInstituteofAcademyofMilitaryMedicalSciencesofChinesePLAfromJunetoOctober2006.Neonatalratsweresacrificedundersterileconditiontoobtainskeletalmusclesoflimbs,whichwerewashedwithcoldPBS(containingbenzylpenicillinandestreptomicina),andmusculartissuewasshearedintopieces.Then,thosemuscularpieceswereaddedwithmixeddigestiveenzyme(containing2g/LcollagenaseⅡ+5g/LdispaseⅡ+0.28g/LCaCl2)astwicevolumeaspieces,dealtwithmechanicalpipettingfor5minutesandculturedinCO2incubatorfor10minutes.Theoperationwasdoneforthreetimesandthemuscularpiecesweredigestedfor45minutesintotal.Moreover,cellsweresuspendedagaininordertoobtainmyoblastsfromskeletalmuscleofneonatalrats.Inaddition,myoblastswerepurifiedwithdifferentialattachmenttechniqueandenzymedigestionsoastoobservemorpho