心血管系统与消化系统蛋白质组学研究进展.ppt

动物模型的制作01自噬形态学的电镜观察02蛋白的提取03双相电泳分离04MALDITOFMS分析05数据库搜索06差异表达蛋白的WB验证与定位研究07Chronicallyinstrumentedpigswerestudiedwithrepetitivemyocardialischemiaproducedbyone,three,orsixepisodesof90minofcoronarystenosis(冠状动脉狭窄)[30%reductioninbaselinecoronaryflowfollowedbyreperfusion(再灌注)every12h]withthenon-ISregionascontrol.Inthismodel,wallthickeningintheISregionwaschronicallydepressedby≈37%.Autophagicvacuoles(Avs,自体吞噬泡)containingremnantsofmitochondriaaredemonstrated.DoublemembraneAVscontainingrecognizablecytoplasmiccontentsaredisplayed.AVscontainingmultivesicularbodiessurroundedbyasequesteringmembranearedemonstrated.12345NI.ArrowsindicateAVs.(Magnifications:1,400–2,000,A–D;5,000,AandBInsets.)TheseAVswerenotobservedinthesix-episode”01040203(Upper)2DgelmapofproteinextractsfromchronicallyISregionaftersixepisodes.(Lower)AmagnifiedgelregionfromtheNIregion(Left)andtheISaftereitherthree(Center)orsix(Right)episodesofCS.ArrowsindicatethespotsaftersixepisodesthatwerealllateridentifiedascathepsinD.MW,molecularweight.Eachlowerimageisa6-foldmagnificationfromtheoriginalgel.ComparisonofexpressionlevelofcathepsinBandDinchronicallyISmyocardiumatdifferentepisodes.(AandB)WesternblotanalysisofcathepsinD(A)andcathepsinB(B)atsixepisodeschronicallyISvs.NIregions(Upper)andthreeepisodes(Lower).TheexpressionofcathepsinsBandDsignificantlyincreasedatsixepisodes(**,P0.01;*,P0.05vs.NI).(C)ThedirectcomparisonoflevelsofcathepsinB(CB)andcathepsinD(CD)inchronicallyISinone,three,andsixepisodes,againshowingmarkedincreasedexpressionofbothproteinsaftersixepisodes.(D)TheelevatedexpressionofcathepsinsBandDwasconfirmedtooccurinmyocytesfromthechronicallyISmyocardium.ADU,arbitrarydensitometricunits.Enzym