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△212000802200080A1stearoyl-CoAdesaturase1,SCD1。30SD。8w16w24wRT-PCRSCD1serinepalmitoyltransferase,SPTmRNATUNEL。HE8w。RT-PCRSCD1mRNASPTmRNA。TUNEL。SCD1SPTAISPTAI。SCD1SPT。A-1R332,R322.61A1673-6273200902-216-04TheCorrelationBetweenSCD1ExpressionandtheApoptosisofRatHepatocytesCausedbyFat-richDietCAIDe-feng1,FANJian-gao2,LUYuan-shan1,2△,CAIXiao-bo21DepartmentofLaboratory,TheFirstAffiliatedPeople'sHospitalofShanghaiJiaotongUniversity,Shanghai,200080,China;2TheDiagnosisandTreatmentCenterofFattyLiver,TheFirstAffiliatedPeople'sHospitalofShanghaiJiaotongUniversity,Shanghai,200080,ChinaABSTRACTObjective:Toinvestigatethecorrelationbetweenstearoyl-CoAdesaturase1expressionandtheapoptosisofrathepa-tocytescausedbyfat-richdiet.Methods:ThirtySpragueDawleyratswererandomlydividedintocontrolandexperimentalgroups.Theratsinexperimentalgroupwerefedwithfat-richdietandthoseincontrolgroupratswerefedwithordinarydiet.Theratsweresacrificedat8w,16w,and24w,respectively.Thedegreeofliverhistologicalchangewasevaluatedbymicroscope.TheratioofliverSCD-1mRNA/β-actinandSPT/β-actinmRNAwereanalyzedbythemethodofRT-PCR.TheapoptosisofrathepatocyteswereanalyzedbyTUNELmethod.Results:Thehepatocytes,stainedwithHE,offat-richgroupwerefilledwithfatundermicroscopyandmetthestandardoffattyliverdiagnosesat8thweek.Fat-richdietmaydecreasetheexpressionofhepaticSCD-1mRNA.LowerlevelofSCD-1mRNAmayresultinexcessivefatdepositinginliverandformingnonalcoholicfattyliver.RT-PCRanalysisshowedthatexperimentalgroupSCD1mRNAexpressionwaslowerthanthecontrolgroup,theexpressionofSPTmRNAincreasedobviously.TUNELapoptosisdetectionshowedthatthelongerfedwithhigh-fatdiet,themoreseriousapoptosisofhepatocytesoccurred.TheleverofSCD1expressionwasnegativelycorre-latedwiththeleverofSPTandapoptosisindex(AI),TheleverofSPTexpressionwaspositivelycorrelatedwithAI.Conclusions:Long-termfat-richdietmaydecreasetheexpressionofhepaticSCD-1mRNA,whichpromotesapoptosiskeygeneSPTexpressionrisingandhepatocytesapoptosisincreasing.Keywords:Fat-RichDiet;Stearoyl-CoaDesaturase-1;SerinePalmitoylTransferase;ApoptosisChineseLibraryClassification:R332,R322.61Documentcode:AArticleID:1673-6273200902-216-041979-。△13386259569E-mail:luyuanshan@126.com2008-07-182008-08-18A1stearoyl-CoAdesaturase-1SCD1。NADHb5、b5SCD3。SCD1CoACoACoA、TG、CE、2,3ADG[1]。β[2]serinepalmitoyltransferaseSPTmRNASPT[2]。freefattyacid,FFA[3]FFA。SCD1216··:controlgroup,H:experimentgroup,aP0.05,bP0.01vscontrolgroup.。11.1SD145g135-155g、。TrizolRNAGibcoBRLPCRFermentasTUNEL。DYY-11TanonEppendorfSZX16ATC201PCR。1.21.2.130215。2%、10%88%。8w16w24w5。14%。1.2.2RNART-PCR0.1gEP1mLTRIzol4℃12000g10min。EP0.2mL15s4℃12000g15EP。0.5mL2min4℃12000g10min。75%1mL10min。4℃7500r/min5min50μLDE-PC。1μLRNADEPC100μlRNA。RNA2μgOligodT18Primer1μLDE-PC12μL70℃5min5×buffer4μLRibolockTMRibonucleaseinhibitor1μL10mMdNTP2μL37℃5minRevertAidTMM-Mulv1μL42℃1h70℃10min。cDNA-80℃。2×PCRMasterMix12.5μL1μM1μLcDNA1μL25μL。SCD1PCR95℃3min95℃20s、56℃30s72℃30s35。SPTPCR94℃3min94℃40s、48℃45s72℃1min32。β-actin5'-AACCCTAAGGCCAA-CCGTGAAAAG-3'5'-TCATGAGTAGTCTGTCAGGT-3'241bpSCD-15'-TGCTGATGTGCTTCATC-CTG-3'5'-GGGAAACCAGGATATTCTCC-3'201bpSPT5'-GGTGTGGCTGTGCTTGAATA-3'5'-AACC-CTCTTCCCAAAACTGA-3'550bp。1.2.3TUNEL1。PBS2K25℃25minPBS23%H2O220℃10minPBS2。250μlTdT37℃60minPBS350μLStreptavidin-HRP30minPBS350-100μLDAB10minPBS。3。。4AI101000。。AI=/×100%。1.3SPSS13.0tSpearmanα=0.05P0.05。22.18w16w24w、1。、。HE8W。16w。24w。1、、SCD1/β-actin、SPT/β-actinmRNAAI(mean±SD,n=5)Table1Bodyweight,thewetliver’squality,theratioofSCD/β-actinandSPT/β-actinmRNAandAIExperimentgroupingWeight(g)Thewetliver’squality(g)TheratioofSCD/β-actinTheratioofSPT/β-actinAIN8w330.45±13.2811.59±0.680.56±0.320.46±0.211.28±0.75H8w389.00±28.27b14.98±2.61a0.38±0.16b0.65±0.25a2.36±0.64aN16w451.54±40.4813.80±1.530.61±0.310.49±0.191.34±0.64H16w512.36±25.40b15.96±2.58a0.32±0.16a0.83±0.23a3.24±1.85aN24w485.48±42.9114.52±1.450.59±0.210.45±0.171.30±0.81H24w558.65±37.38a17.90±1.93a0.29±0.24a1.03±0.29b4.56±2.01b217··。SCD1mRNASPTmRNASpearman-0.43P0.012。SCD1SPT12。2.3TUNELTUNEL。TUNEL。、。3。SCD1mRNAAISpearman-0.48P0.05SPTmRNAAISpearman0.63P0.052。3、。8w16w24wSCD1mRNASCD1mRNA8w。Cohenetal[4]SCD1SREBP[5][6]neuropeptideYNPYSCD1。SCD1mRNA[7]、、[8]。SPTmRNATUNELAI。SCD1mRNASPTmRNAAISPTmRNAAI。SCD1。SCD1AAAA。SCD1FFAAASPTSPT[9]。Parketal[10]SPT。[11]。ShahCetal[12]C57BL/6JSPTC16C18C18:1。[13]A-[14]。Akt[15-16]。SCD1A[14]。。、[17]。、、A、[18]。ROS[13]、2SCD1/β-actin、SPT/β-actinAITable2ThecorrelationamongSCD1/β-actin,SPT/β-actinandAIGroupSCD1/β-actinvsSPT/β-actinSPT/β-actinvsAISCD1/β-actinvsAIControlgroup151515Experimentgroup151515-0.430.63-0.480.010.010.011SCD1MMarkerSSCD1ββ-actinN8w、16w、24wFig.1SCD1electrophoretogram,MisMarker,SisSCD1,βisβ-actin,Niscontrolgroup,8w,16w,24wisexperimentgroup2SPTMMarkerN8w、16w、24wFig.2TheSPTelectrophoretogram,MisMarker,Niscontrolgroup,8w,16w,24wisexperim