IKIP通過靶向IKKβ負(fù)向調(diào)節(jié)NF-κB介導(dǎo)的炎性細(xì)胞因子的表達(dá)的開題報告

IKIP通過靶向IKKβ負(fù)向調(diào)節(jié)NF-κB介導(dǎo)的炎性細(xì)胞因子的表達(dá)的開題報告IntroductionInflammationisafundamentalresponseoftheinnateimmunesystemagainsttissuedamage,pathogeninvasion,andotherharmfulstimuli.Theactivationofinflammatorysignalingpathwaysleadstotheproductionandreleaseofcytokines,chemokines,andothereffectormoleculesthatrecruitandactivateimmunecells.ThenuclearfactorkappaB(NF-κB)isacriticaltranscriptionfactorthatregulatestheexpressionofmultiplegenesinvolvedininflammation,immunity,cellsurvival,andproliferation.ThedysregulationofNF-κBsignalingisimplicatedinthepathophysiologyofvariousinflammatorydiseases,includingrheumatoidarthritis,inflammatoryboweldisease,psoriasis,andsepsis.TheactivationofNF-κBinvolvesthesignal-induceddegradationofIκBα,theinhibitorofNF-κB,andthenucleartranslocationoftheNF-κBdimers,includingp65/p50,p65/c-Rel,andRelB/p50.TheactivationoftheIκBkinase(IKK)complex,composedofIKKα,IKKβ,andNEMO,isacriticalstepinthisprocess.IKKβisconsideredtheprimarykinasethatphosphorylatesIκBα,leadingtoitsubiquitinationandsubsequentproteasomaldegradation.IKIP,alsoknownastheinhibitorofkappaBphosphorylation,isasmallproteinthatbindstotheIKKcomplexandinhibitsitsactivity.IKIPhasbeenshowntonegativelyregulatetheactivationofNF-κBandtheexpressionofNF-κBtargetgenes,includingTNFα,IL-1β,COX-2,andiNOS.IKIPexpressionlevelsaredecreasedininflamedtissuesfrompatientswithinflammatorydiseases,suggestingthatIKIPdeficiencymaycontributetoNF-κBhyperactivationandinflammation.WehypothesizethattheoverexpressionofIKIPinimmunecellscouldattenuateNF-κBactivationandtheexpressionofpro-inflammatorycytokines,leadingtoareducedinflammatoryresponse.Inthisproject,weaimtoinvestigatetheeffectofIKIPoverexpressiononNF-κBsignaling,theexpressionofinflammatorycytokines,andthephenotypeofimmunecells.Objectives1.ToconstructanexpressionvectorforIKIPoverexpressioninimmunecells2.ToconfirmtheoverexpressionofIKIPinimmunecellsbyWesternblotandqPCR3.ToassesstheeffectofIKIPoverexpressiononNF-κBactivationinimmunecellsbyluciferasereporterassay4.Tomeasuretheexpressionofpro-inflammatorycytokines,suchasTNFα,IL-1β,andIL-6,inimmunecellsuponIKIPoverexpressionbyELISAandqPCR5.ToexaminethephenotypeofimmunecellsuponIKIPoverexpressionbyflowcytometryanalysisMethods1.ConstructionoftheIKIPexpressionvectorThehumanIKIPcDNAwillbeclonedintothepCDH-CMV-MCS-EF1-Purolentiviralvector.Thevectorwillbeverifiedbyrestrictionenzymedigestionandsequencing.2.CellcultureandtransfectionTheTHP-1humanmonocyticcelllinewillbeculturedinRPMI1640mediumsupplementedwith10%fetalbovineserum,100U/mLpenicillin,and100μg/mLstreptomycin.TooverexpressIKIP,THP-1cellswillbetransducedwiththeIKIPlentiviralvectorusingpolybrene.Thecellswillbeselectedwithpuromycintogeneratestablecelllines.3.WesternblotandqPCRTheproteinandRNAsampleswillbepreparedfromtheTHP-1cells,andtheexpressionofIKIPwillbedeterminedbyWesternblotandqPCR.TheexpressionofNF-κBcomponentsandtargetgeneswillalsobeanalyzed.4.LuciferasereporterassayANF-κBluciferasereporterplasmidwillbetransfectedintotheTHP-1cells.FollowingstimulationwithLPS,theluciferaseactivityinthecellswillbemeasuredtoevaluatetheeffectofIKIPoverexpressiononNF-κBactivation.5.ELISAandqPCRTheexpressionofTNFα,IL-1β,andIL-6willbemeasuredintheculturesupernatantsandcelllysatesoftheTHP-1cellsbyELISAandqPCR.6.FlowcytometryThephenotypeoftheTHP-1cellsuponIKIPoverexpressionwillbeassessedbyflowcytometryanalysisofcellsurfacemarkers,suchasCD14,CD11b,CD16,andHLA-DR.ExpectedResultsWeexpectthattheoverexpressionofIKIPinTHP-1cellswilldownregulateNF-κBactivationandreducetheexpressionofpro-inflammatorycytokines,suchasTNFα,IL-1β,andIL-6.WealsoanticipatethatIKIPoverexpressionwillalterthephenotypeofTHP-1cells,potent