简介：AIMTodeterminetheroleofcorticotropinreleasingfactorreceptor(CRF2)inepithelialpermeabilityandenterocytecelldifferentiation.METHODSForthispurpose,weusedratSpragueDawleyandvariouscoloncarcinomacelllines(SW620,HCT8R,HT-29andCaco-2celllines).ExpressionofCRF2proteinwasanalyzedbyfluorescentimmunolabelinginnormalratcolonandthenbywesternblotindissociatedcolonicepithelialcellsandinthelysatesofcoloncarcinomacelllinesorduringtheearlydifferentiationofHT-29cells(tenfirstdays).ToassesstheimpactofCRF2signalingoncoloniccelldifferentiation,HT-29andCaco-2cellswereexposedtoUrocortin3recombinantproteins(Ucn3,100nmol/L).Insomeexperiments,cellswerepre-exposedtotheastressin2b(A2b)aCRF2antagonistinordertoinhibittheactionofUcn3.Intestinalcelldifferentiationwasfirstanalyzedbyfunctionalassays:thetrans-cellularpermeabilityandthepara-cellularpermeabilityweredeterminedbyDextran-FITCintakeandmeasureofthetransepithelialelectricalresistancerespectively.Morphologicalmodificationsassociatedtoepithelialdysfunctionwereanalyzedbyconfocalmicroscopyafterfluorescentlabelingofactin(phaloidin-TRITC)andintercellularadhesionproteinssuchasE-cadherin,p120ctn,occludinandZO-1.Theestablishmentofmatureadherensjunctions(AJ)wasmonitoredbyfollowingthedistributionofAJproteinsinlipidraftfractions,afterseparationofcelllysatesonsucrosegradients.Finally,themRNAandtheproteinexpressionlevelsofcharacteristicmarkersofintestinalepithelialcell(IEC)differentiationsuchasthetranscriptionalfactorkrüppel-likefactor4(KLF4)orthedipeptidylpeptidaseIV(DPPIV)wereperformedbyRT-PCRandwesternblotrespectively.ThespecificactivitiesofDPPIVandalkalinephosphatase(AP)enzymesweredeterminedbyacolorimetricmethod.RESULTSCRF2proteinispreferentiallyexpressedinundifferentiatedepithelialcellsfromthecryptsofcolonandinhumancoloncarcinoma

简介：AIM:Todeterminethealterationsinratenterocytemitochondrialrespiratoryfunctionandenzymeactivitiesfollowingtraumaticbraininjury(TBI).METHODS:Fifty-sixmaleSDratswererandomlydividedintosevengroups(8ratsineachgroup):acontrolgroup(ratswithshamoperation)andtraumaticbraininjurygroupsat6,12,24h,days2,3,and7afteroperation.TBImodelswereinducedbyFeendy’sfree-fallingmethod.Mitochondrialrespiratoryfunction(respiratorycontrolratioandADP/Oratio)wasmeasuredwithaClarkoxygenelectrode.TheactivitiesofrespiratorychaincomplexⅠ-Ⅳandrelatedenzymesweredeterminedbyspectrophotometry.RESULTS:Comparedwiththecontrolgroup,themitochondrialrespiratorycontrolratio(RCR)declinedat6handremainedatalowleveluntilday7afterTBI(control,5.42±0.46;6h,5.20±0.18;12h,4.55±0.35;24h,3.75±0.22;2d,4.12±0.53;3d,3.45±0.41;7d,5.23±0.24,P<0.01).Thevalueofphosphate-to-oxygen(P/O)significantlydecreasedat12,24h,day2andday3,respectively(12h,3.30±0.10;24h,2.61±0.21;2d,2.95±0.18;3d,2.76±0.09,P<0.01)comparedwiththecontrolgroup(3.46±0.12).Twotroughsofmitochondrialrespiratoryfunctionwereseenat24handday3afterTBI.TheactivitiesofmitochondrialcomplexⅠ(6h:110±10,12h:115±12,24h:85±9,day2:80±15,day3:65±16,P<0.01)andcomplexⅡ(6h:105±8,12h:110±92,24h:80±10,day2:76±8,day3:68±12,P<0.01)wereincreasedat6hand12hfollowingTBI,andthensignificantlydecreasedat24h,day2andday3,respectively.However,therewerenodifferencesincomplexⅠandⅡactivitiesbetweenthecontrolandTBIgroups.Furthermore,pyruvatedehydrogenase(PDH)activitywassignificantlydecreasedat6handcontinuedupto7dafterTBIcomparedwiththecontrolgroup(6h:90±8,12h:85±10,24h:65±12,day2:60±9,day3:55±6,day7:88±11,P<0.01).Thechangesinα-ketoglutaricdehydrogenase(KGDH)activityweresimilartoPDH,exceptthatthedecreaseinKGDHactivitybeganat12hafterTBI(12h:90±12,24h:80±9,day2:76±15,day3:68±7,