斑马鱼模型的心肌病相关致病基因myl的功能研究

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1、上海交通大学医学院博士学位论文zebrafishasamodelorganismtoidentifyELCandRLCorthologues，respectively,andfurthermore，characterizedtheirfunctionsduringcardiogenesisandgetimplicationofELCsandRLCsinpathogenesisofcardiomyopathy．MethodsThehumanMYIApeptidesequencewasusedtosearchtheN

2、CBIdatabase，andanalysisofthenucleotideandaminoacidsequencesforthezebrafishandhumanorthologueswereperformedbyClustalWandDSGene．Whole-mountinsituhybridizationWasperformedtodetectgenesexpression．Morpholinoantisenseoligonucleotides(morpholinos)wereinjectedtoknock

3、downthegenes．MoviesofbeatingheartswererecordedusingaZeissmicroscopeequippedwithaNikoncamera．Imagesfrommovieswerethenusedtocountheartrate(HR)andmeasureshorteningfraction(SF)，enddiastolicvolume(EDV)andendsystolicvolume(ESV)．Todetectsarcomereassemblyandmeasuresa

4、rcomerelengthandcardiomyocytessize,whole-mountimmunofluorescencestainingWasperformedandimagedusinganAxioplanllZeissmicroscopeequippedwithanApotome．ThecardiomyocytesnumberwascountedafterprocessingtheimagesfromTg(cmlc2：nuDsRed)transgenicline．CelldeathWasanalyze

5、dbyTUNELassay．上海交通大学医学院博士学位论文ResultsWeidentifiedcmlclisthemaincardiachomologueofELCandcmlc2isthemaincardiachomologueofRLCinzebrafishbybioinformaticsanalysis．Depletionofeithercmlclorcmlc2usingmorpholino—modifiedantisenseoligonucleotidesleadstoadisruptioninsarc

6、omerestructureaswellascompromisescardiacfunction，althoughthroughseeminglydistinctmechanisms．Whilemyosinstillassemblesintoarod—likestructureinbothmorphants，thesarcomerelengthislongerincmlclmorphantsbutshorterincmlc2morphantsthanthatinwildtypeembryos．Inaddition

7、，cardiomyocytesizeandnumberareincreasedupondepletionofcmlc1，resultinginalargerventricularchambervolume，whereasdepletionofcmlc2leadstoareductionincardiomyocytesizeandnumber．Thesedataindicatedthatcmlclandcmlc2arerequiredforcardiogenesisandmayhavedistinctfunctio

8、ns．ConclusionsOurdataelucidateddistinctrolesforcmlclandcmlc2duringzebrafishcardiogenesis，suggestingthatcardiomyopathiesresultingfromhumanmutationsinELCsversusRLCsmayhavedistinctpathologic