MicroRNA-122及其靶基因CPEB1在角膜移植排斥中的作用研究.pdf

1、分類號：R772.2密級：不保密UDC：617學校代碼：11065碩士學位論文MicrNA122及其靶基因CPEB1在角膜移植排斥中的作用研究MicrNA122及其靶基因CPEB1在角膜移植排斥中的作用研究李鳳潔指導教師史偉云教授學科專業(yè)名稱眼科學論文答辯日期2015年05月22日TheRoleofMicrNA122ItsTargetGeneCPEB1InCnealAllograftRejectionAbstractPurposes：T

2、oinvestigatetheroleofmicrNA122itstargetgeneCPEB1duringperatingconeraltransplantationrejection.Methods:1.Establishingmiceperatingkeratoplastymodel2.RealtimePCRwasusedtoanalyzetheexpressionofmiR122CPEB1inthreelayersofcneat

3、issueimmunecells.3.Overexpressionagomirantagomir122analyzingCPEB1expressionapoptosis.4.CnealstromalcelllinewasculturedinfectedwithlentiviruscontaingmiR122bindingsitewildtypemutatedCPEB1.Cellswerethentreatedwithinflammaty

4、cytokines(TNFαIL1βTNFγ)f48hours.ApoptosisofcnealstromalcellsNOgenerationwasdeterminedusingannexinVstainingNOkit.Results:1.Detectionofthepercentageofimmunecellsbyflowcytometryafterperatingcnealtransplantation:thepercentag

5、eofimmunecellsissignificantlyhigherinthenonrejectiongroup(P0.05).2.RealtimePCRanalysis：theexpressionofbothmiR122CPEB1issignificantlyhigherinthecnealstromallayercomparedwithstromallayerendodermislayer（P0.0001)miR122wasnot

6、expressedintheimmunecells.3.ThestromalcellswereinfectedwithlentiviruscontaingmiR122bindingsitewildtypemutatedCPEB1.Theinfectionefficiencywasmethan90%.4.Overexpressionagomirantgomir122analysis:Transfectionagomir122theexpr

7、essionofCPEB1islowerthancontrolreducedapoptosistransfectionantagomir122theexpressionofCPEB1ishigherthancontrolincreasedapoptosis(P0.05).5.ApoptosisNOdetection:overexpressionofCPEB1promotecnealstromalcellsapoptosisNOgener

8、ation(P0.05)Allsamplestreatedwithinflammatycytokinesshowedincreasedapoptosis(P0.05)；AftertreatedwithcytokinethestromalcellsexpressingmiR122bindingsitemutatedCPEB1showedsignificantlyhigherrateofapoptosiscomparingwithstrom

9、alcellsexpressingmiR122bindingsitewildtypeCPEB1（P0.05）TheapoptosisissimilarbetweenunusedcytokinetreatedstromalcellsexpressingmiR122bindingsitewildtypemutatedCPEB1Regardlessofcytokinestimulationthestromalcellsexpressingmi

10、R122bindingsitemutatedCPEB1showedsignificantlyhigherrateofNOgenerationcomparingwithstromalcellsexpressingmiR122bindingsitewildtypeCPEB1（P0.05）.Conclusions:miR122CPEB1werehighestexpressedinthecnealstromallayerexpressionof

11、miR122wasnotdetectedintheimmunecellsWhentheexpressionofmiR122wasdownregulatedtheexpressionofitstargetCPEB1wasincreased.ThisleadstoincreasedapoptosisNOgenerationofcnealstromalcellspromotescnealtransplantationrejection.Pos