上皮細胞生理學前沿 (2)課件

上皮細胞生理學前沿20141106高東東ContentsBackgroundAbouttheauthorEmail:()Web:BackgroundAboutthecornea角膜(Cornea)是眼睛最前面的透明部分，覆蓋虹膜、瞳孔及前房，并為眼睛提供大部分屈光力。加上晶體的屈光力，光線便可準確地聚焦在視網膜上構成影像。為了保持透明，角膜并沒有血管，透過淚液及房水獲取養(yǎng)份及氧氣。BackgroundAboutthecorneaepithelium角膜上皮厚約50微米，由5-6層鱗狀上皮細胞組成，無角化，排列特別整齊，易與Bowman層相分離.其共有3種類型細胞：基底細胞、翼狀細胞、扁平細胞。角膜上皮細胞約每周更換一次。Background角膜緣干細胞(LSCs)

是角膜上皮細胞再生的來源。對于保持角膜透明和維持正常的生理功能極為重要。AbouttheLSCsLimbus（異色邊緣）BackgroundAboutcornealsurfacediseaseDeficiencyinLSCsorcornealepithelium—whichturnscornea

intoanon-transparent,keratinized

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

ectodermal

tissues.BackgroundMethods★Genome-widegeneexpressionmicroarrayanddataanalysis

該方法首先從每個mRNA的3’端酶切得到一段21bp的TAG片段（特異性標記該基因）；然后通過高通量測序，得到大量的TAG序列，不同的TAG序列的數量就代表了相應基因的表達量；通過生物信息學分析得到TAG代表的基因、基因表達水平、以及樣品間基因表達差異等信息?！颮NA-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