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1、天津医科大学硕士学位论文AbstractObjective:Themechanismofmolecularbiologyofintervertebraldiscdegeneration(IVDD)stillkeepmagic.Humannucleuspulposuscell(HNPC)apoptosisplaysanimportantroleinthedevelopmentofintervertebraldiscdegeneration(IVDD).TNF-caninducenucleuspulposus
2、cellsapoptosis,whichhasanintimaterelationshipwithIVDD.Theextrinsicfactorcancombinewithcellsurfacereceptor,andproducebiologicaleffectthroughendogenousfactor,forexamplemiRNAs.OurpreviousresearchrevealedthatamongallofthedysregulatedmicroRNAsinthedegeneratednu
3、cleuspulposustissuesofpatientwithIVDD,miR-494(miR-494)isthemostsignificantlyincreased.However,theinfluenceofmiR-494onTNF--inducedHNPCapoptosishasnotbeenconfirmed.ThisstudywasdesignedtoevaluatetheeffectofmiR-494ontheHNPCapoptosisinducedbyTNF-αandtoexploret
4、hepossiblemechanismofthisprocess.Methods:Collecttheclinicaldataandnucleuspulposusofspineburstfracturepatients.Accordingtopreoperativeimagingdata(X-ray,CT,MRI),weevaluatethesituationofpatientsbyTLISSscoresystemandloadsharingscores.AccordingtotheMRI,thedegre
5、eofdegenerationofIVDDisevaluatedbyPfirrmannclassification.Weusedenzymedigestionmethodtoobtainprimarynucleuspulposuscells,andappraisednucleuspulposuscells.HNPCswerestimulatedwithTNF-αatdifferentconcentrations(0ng/ml,10ng/ml,50ng/ml,or100ng/ml)for0h,8h,16h,o
6、r24h.AnnexinV-PE/7-AADassaysandreal-timequantitativePCRwereusedtodetectthecellapoptosisratesandmiR-494expression.Second,wesuccessfullyknockeddownendogenousmiR-494inHNPCsvialentiviralantigomiR-494vectorinfectionandthenstimulatedwithTNF-α(100ng/ml,16h).Thera
7、tesofapoptosisandmiR-494expressionwerethendetectedagain.Additionally,adual-luciferasereporterassayandwesternblottingwereusedtodeterminewhetherJunDisatargetofmiR-494.Finally,westernblottingwasusedtoanalyzetheexpressionofcytochromeC.Results:Wefoundthattherat
8、eofapoptosisincreasedwithconcentration,time(p<0.05)andmiR-494expression(p<0.05).Therateofapoptosisinthe100ng/ml,16hgroupappearedtobesuitable.Aftertransfection,theapoptosisrateandmiR-494III万方数据天津医科大学硕士学位论文expr
