上皮细胞生理学前沿 (2)课件

上皮细胞生理学前沿20141106高东东ContentsBackgroundAbouttheauthorEmail:()Web:BackgroundAboutthecornea角膜(Cornea)是眼睛最前面的透明部分，覆盖虹膜、瞳孔及前房，并为眼睛提供大部分屈光力。加上晶体的屈光力，光线便可准确地聚焦在视网膜上构成影像。为了保持透明，角膜并没有血管，透过泪液及房水获取养份及氧气。BackgroundAboutthecorneaepithelium角膜上皮厚约50微米，由5-6层鳞状上皮细胞组成，无角化，排列特别整齐，易与Bowman层相分离.其共有3种类型细胞：基底细胞、翼状细胞、扁平细胞。角膜上皮细胞约每周更换一次。Background角膜缘干细胞(LSCs)

是角膜上皮细胞再生的来源。对于保持角膜透明和维持正常的生理功能极为重要。AbouttheLSCsLimbus（异色边缘）BackgroundAboutcornealsurfacediseaseDeficiencyinLSCsorcornealepithelium—whichturnscornea

intoanon-transparent,keratinized

skin-likeepitheliumcausescornealsurfacediseasethatleadstoblindnessinmillionsofpeopleworldwideBackgroundAboutthePAX6★Pax6isatranscriptionfactorpresentduringembryonicdevelopment.★Itactsasa"mastercontrol"geneforthedevelopmentofeyesandothersensoryorgans,certainneuralandepidermaltissuesaswellashomologousstructures,usuallyderivedfrom

ectodermal

tissues.BackgroundMethods★Genome-widegeneexpressionmicroarrayanddataanalysis

该方法首先从每个mRNA的3’端酶切得到一段21bp的TAG片段（特异性标记该基因）；然后通过高通量测序，得到大量的TAG序列，不同的TAG序列的数量就代表了相应基因的表达量；通过生物信息学分析得到TAG代表的基因、基因表达水平、以及样品间基因表达差异等信息。★RNA-seqandhierarchicalclusteranalysis★LentiviralRNAinterferenceandPAX6transduction★Immunofluorescenceandlaserconfocalmicroscopy★QuantitativePCR★Westernblotanalysisandco-immunoprecipitation★Celltransplantation

StrategiesGeneknockdownLSCengineeredexpressionofPAX6CelltransplantationsuccessfullyrepairedcornealepitheliumdefectWNT7AandPAX6arekeyfactorsLSCCECgenome-widegeneexpressionanalysisWNT7APAX6LSC/CECSESCResults

NormaldifferentiationFig1K19-LSCK3/K12-CECP63-stemcellResults

NormaldifferentiationEDFig.1

P63-stemcellFig1K1/k10-SECK19-LSCsK12-CECResults

AbnormalepidermaldifferentiationFig1PathologicalconversionofCECsintoskin-likeepithelialcells,asindicatedbymorphologicalchangesandswitchesinkeratinexpression

What’sthekeyfactors?K3/K12-CECK1/K10-SEC

K5-stemcellResults

ThekeyfactorsFig2▼feeder-freeLSCscultureandthree-dimensionalLSCdifferentiationK5,K14,P63-stemcellK3/K12-CECK1/K10-SECfeeder-freeLSCsculturethree-dimensionalLSCdifferentiationResults

ThekeyfactorsExtendedDataFig2WNT7AandPAX6werehighlyexpressedinLSCsandCECswhencomparedtoSESCs▼qPCRanalysisofWNT7AandPAX6expressioninLSCsandCECscomparedtoSESCsResults

ThekeyfactorFig3TheclinicalrelevanceofWNT7AandPAX6expressioninLSCsandCECs

▼ImmunofluorescenceofthepathologicalsectionP63-stemcellK3/K12-CECResults

AreWNT7Aandpax6thekeyfactors?

LSCCEC？WNT7AWNT7A-WNT7A？Results

ValidationofthekeyfactorEDFig4Geneknockdown▼PhasecontrastphotographsshowingeffectsofWNT7AandPAX6knockdownsResults

Validationofthekeyfactor▼GeneexpressionprofilingFig4

Together,thesedatasuggestthatdefectsintheWNT7A–PAX6axisarelikelytoberesponsibleformetaplasticconversionofcornealcellstoskinepidermal-likecellsincornealdiseasesinhuman

WNT7ALSCCECWNT7A-How

dotheWNT7Aandpax6function？ResultsWNT7Aandpax6arethekeyfactorsResults

The

keyfactors’signalpathwayFig3WeguessthatWNT7AactsupstreamofPAX6duringCECdifferentiation.EDFig4WNT7A▼QuantitativePCRanalysisandwesternblotanalysisResults

The

keyfactors’signalpathwayFZD5WNT7ALSCCECWNT7A-？Results

Fig3ImportantroleofPAX6▼ImmunofluorescencestainingK3/K12-CECK19-LSCP63-stemcellResults

Fig4ImportantroleofPAX6▼QPCRanalysisofgeneexpressionofkeratinsinPAX61SESCsResults

Fig4ImportantroleofPAX6▼GeneexpressionprofilingLSCCECResults

FZD5WNT7ALSCCECSWNT7A-Results

Fig5TreatmentandrepairpotentialofSESCswithengineeredexpressionofPAX6GFP-labelledPAX6+SESCstransplantationtothecorneaofarabbitlimbalstemcelldeficiencymodelK3/K12-CECK10-SECSESCLSCResults

Fig32monthsposttransplantationH&Estainwhitelightmicrographslit-lampmicrographfluoresceindyestainingNormalcornea

GFP-labelledLSCsGFP-labelledPAX6+SESCs

GFP-labelled,shPAX6-treatedLSCsamnioticmembraneResults

Fig5Rabbitcornea3monthsposttransplantationwithGFP-labelledPAX6+SESCs:smooth,transparentcorneawithpositiveGFPsignalsTogether,thesedatademonstratethatSESCswithPAX6expressionareableto