牛LXRα基因mRNA重组慢病毒载体的构建及其对牛肌肉卫星细胞的干扰效果.pdf 10页

'中国科技论文在线http://www.paper.edu.cnConstructionofRecombinantLentivirusVectorTargetingBovineLXRαmRNAandItsSilencingEffectsonBovine#5MuscleSatelliteCells*LiuYongfeng,ZhaoLu,KuTing,YangXingbin(CollegeofFoodEngineeringandNutritionalScience,ShaanxiNormalUniversity,Xi"an710062)Abstract:TheliverXreceptorα(LXRα)isamemberofthenuclearhormonereceptorsuperfamilywhichcouldregulatethetranscriptionofthegenesinvolvedincholesteroltransportation.Recent10studiesshowedthatLXRαisconsideredasacriticalregulatorincholesterolhomeostasisinmacrophages,whichcouldregulateseveralgenesinvolvedincholesteroltransport,suchastheATP-bindingcassettetrans-porters(ABCs),ABCA1,ABCG1,apolipoproteinE(ApoE)andlipoproteinlipase(LPL).Inourstudy,fourpairsofinhibitionshRNAweredesignedtoexclusivelytargetbovineLXRαmRNA.LentiviralvectorcontainingLXRαshRNAswereconstructedthroughBP15andLPrecombinationsystem,andusedtoobtainedthecorrespondinglentivirusesin293Tcells.ThetitersoftheLenti-14MR0054-01,-2,-3and-04viruseswere3×108TU/ml,2×108TU/ml,2.5×108TU/ml,3.5×108TU/ml,respectively.TheknockdownefficiencyofpLenti-03targetingLXRαreached88%inbovinemusclesatellitecells.Furthermore,themRNAexpressionofRXRαwasupregulatedinbovinemusclesatellitecells,whereasthatofPPARα,PPARγ,ABCA1,LPL,ApoE20weredownregulatedat48hpost-pLenti-03virusesinfection.Therefore,theefficientlentivirusvectorinhibitingbovineLXRαexpressionwasobtainedinthepresentstudy,providingbasisforloss-of-functionofLXRαinbovinemusclesatellitecellsKeywords:bovineLXRαgene;lentivirus;vectorconstruction;genesilencing250IntroductionObesity,hypertension,dysmetabolismarediseasescausedbyunhealthydietcustomwhichinfluencedhuman’slivingquality.Thus,alow-cholesterollifestyleisnecessary.Currently,peoplehavepaidmuchattentiontobalancingtheirdietbylow-intakeofcholesterol.Producinggoodqualitymeatwithlow-fat(beef,mutton,pork,etc.)isavitalwaytoprovidepeoplewithhealthymealscondition.30TheliverXreceptorα(LXRα,NR1H3)andLXRβ(NR1H2)aremembersofthenuclearhormonereceptorsuperfamily.LXRαisabundantlyexpressedintheliver,intestine,adiposetissue,kidneyand[1,2]immunemacrophages,whereasLXRβisubiquitouslyexpressed.LiverXreceptors(LXRs)areactivatedinresponsetointracellularlipidaccumulation,whichcouldregulatetranscriptionofanarray[3,ofgenesinvolvedintheregulationofcholesterolhomeostasisandreversecholesteroltransport4]35.CholesterolhomeostasisisintricatelyregulatedbyabatteryoftranscriptionfactorsamongwhichLXRsarenuclearreceptorsthatplayacrucialroleintranscriptionalregulationoflipidmetabolismand[5]inflammation.ActivatedLXRsformaheterodimerwiththeretinoidXreceptorα(RXRα),which[6]bindstoLXRresponsiveelements(LXREs)andconsequentlypromotestargetgeneexpression.LXR/RXRheterodimersarecharacterizedbytheabilitytobeactivatedbyligandinanindependent40manner.Thus,LXR/RXRheterodimersareactivatedbytheRXRligand,e.g.,9-cisretinoicacid,andtheLXRligands,e.g.oxysterols,orareactivatedsynergisticallyinthepresenceofligandsforboth[7]receptors.RecentstudiesshowedthatLXRαisconsideredasacriticalregulatorincholesterolhomeostasisinmacrophages,whichcouldregulateseveralgenesinvolvedincholesteroltransport,suchFoundations:theresearchfundforthedoctoralofhighereducationofChina(20130202120008),theChinapostdoctoralsciencefoundation(2015M570811)andthefundamentalresearchfundsforthecentraluniversitiesofChina(GK201502008).Briefauthorintroduction:LiuYongfeng(1981-),Male,AssociateProfessor,FoodMolecularNutrition.E-mail:yongfeng200@126.com-1- 中国科技论文在线http://www.paper.edu.cnastheATP-bindingcassettetrans-porters(ABCs),ABCA1,ABCG1,apolipoproteinE(ApoE)and[8]45lipoproteinlipase(LPL).[9]ABCA1,ABCG1andApoEarealltargetgenesregulatedbyLXR.Besides,peroxisomeproliferator-activatedreceptor-alpha(PPARα)canactivatethecytochromep450enzymes,resultinginsomeofthehydroxylcholesterolasLXRαendogenousligandfurtheractivateLXRα,whichfurtheractivatingABCA1regulatedcholesterolefflux.RecentstudiesindicatedthatPPARγenhances[10]50cholesteroleffluxbyinducingthetranscriptionofLXRα.PPARγalsoinducestheexpressionofABCA1andABCG1,andpromotescholesteroleffluxfrommacrophagesthroughatranscriptional[11-13]cascademediatedbyLXRα.LXRαcanalsoactivateLPL.Somestudieshaverevealedthatcholesterol-inducedLPLgeneexpressionintheliverisdirectlyregulatedbyRXR/LXRheterodimersin[14]atissue-specificmanner,whichismediatedpredominantlybyLXRαinvivo.55LXRαisimportantforcholesterolmetabolisminhumanandmice.BovineLXRαgenealsocontrolsthecholesterollevelandtheoutflowofcholesteroleffluxinmuscle.GiventhattheimportanceofLXRαgene,regulatingcholesterolinmetabolism,exploringLXRαgenefunctionincholesterolmetabolisminmusclecellstobalancethecholesterolcontentandimprovingthequalityofbeefareofgreatsignificance.PackagingandproliferatinglentiviruswithRNAinterference(RNAi)techniqueisa[15]60conservedbiologicalresponsetodouble-strandedRNA,RNAiisasequence-specificprocessthatregulatesgeneticfunctionsandprovidesdefenseagainstvirusatthepost-transcriptionallevelin[16,17]mammaliancellsandanimals.LentivirusvectorisakindofinactivatedHIVviruswithmanyadvantages,suchashightransfectionrate,stableexpressionintargetcellsandgoodsecurity,soithasbeenwildlyusedintransgenicresearchasacrucialtool.65Lentiviralexpressionvectorisakindoflong-actingsystemwhichcaninfecthostcellswithhigh-efficiency.Thus,weusethismediationsystemtoachievefunctionalgeneresearchonbovinemusclesatelliteprimarycells.ConsideringtheimportanceofLXRαgeneandrelatedstudyonbovinehasnotbeenreportedyet.WehypothesizedthatLXRαmightinfluencesomecholesterolmetabolismregulatinggenes.Toaddressthishypothesis,wesuccessfullyconstructvirusvectorinordertotransfect70bovinecellsanddetectsomecholesterolregulationgenes.Toourknowledge,thisstudyfirstdemonstratedtheinterferenceofLXRαonbovinemusclesatellitecellsinvitrotoinvestigateLXRαgene’sfunctionanditstargetgenes.Furthermore,itcanprovideuswithvaluableinformationforfurtherstudyingofbovineLXRαgenemechanism.1Materialsandmethods75Unlessstatedotherwise,allchemicalsandbiochemicalsusedinthisstudyaretestedforcellcultureandareofmolecularbiologygrade.1.1CellcultureHEK293TCells,thebovinemusclesatellitecellsweregivenbyNBCIC(NationalBeefCattleImprovementCenter,NorthwestA&FUniversity,Yangling,China),andidentifiedwithcellmarkersby80Immunofluoresencestaining,culturedinDulbecco"smodifiedEagle"smedium(DMEM,gibco)withstableL-glutaminesupplementedwith20%fetalbovineserum(FBS,gibco),10%horseserum(HS，gibco),100U/mlpenicillin（sigma）,100µg/mlstreptomycin（sigma）(completegrowthmedium)，-2- 中国科技论文在线http://www.paper.edu.cnincubatedat37°Cunder5%CO2atmosphere（Thermo）.1.2DesignofshRNAsequenceandsystem85ThesequenceofbovineLXRα(NM_001014861.1)inGenBank.InterferenceshRNAsequencesweredesignedononlinesoftware.FourshRNAoligosandtheshRNA-NCsequenceoligowereshownintable1.Tab.1OligonucleotidesusedforRNAiNO.Sequence14MR0054-1-FTGCTGAGAAACATCAGGCACAGGAGCGTTTTGGCCACTGACTGACGCTCCTGTCTGATGTTTCT14MR0054-1-RCCTGAGAAACATCAGACAGGAGCGTCAGTCAGTGGCCAAAACGCTCCTGTGCCTGATGTTTCTC14MR0054-2-FTGCTGTGATAAGACACACTCCTCCCGGTTTTGGCCACTGACTGACCGGGAGGAGTGTCTTATCA14MR0054-2-RCCTGTGATAAGACACTCCTCCCGGTCAGTCAGTGGCCAAAACCGGGAGGAGTGTGTCTTATCAC14MR0054-3-FTGCTGAATCGCAGACGTCTTCAGCAGGTTTTGGCCACTGACTGACCTGCTGAACGTCTGCGATT14MR0054-3-RCCTGAATCGCAGACGTTCAGCAGGTCAGTCAGTGGCCAAAACCTGCTGAAGACGTCTGCGATTC14MR0054-4-FTGCTGAACACTTGCTCTGAGTGGACGGTTTTGGCCACTGACTGACCGTCCACTGAGCAAGTGTT14MR0054-4-RCCTGAACACTTGCTCAGTGGACGGTCAGTCAGTGGCCAAAACCGTCCACTCAGAGCAAGTGTTCNegative-FTGCTGAAATGTACTGCGCGTGGAGACGTTTTGGCCACTGACTGACGTCTCCACGCAGTACATTTNegative-RCCTGAAATGTACTGCGTGGAGACGTCAGTCAGTGGCCAAAACGTCTCCACGCGCAGTACATTTcSyntheticsingle-strandedDNAoligowasdilutedwithddH2O.Then,wemixed0.5μlofeachof90topstrandoligoandbottomstrandoligoand2μl10×oligoannealingbuffer,adding8μlddH2Ouptoaofinalvolumeof20μltoanneal.Oligomixturewasheatedat95Cfor5min,andthenplacedatroomtemperaturefor20minformingdouble-strandedDNA.Syntheticdouble-strandedDNAwasdilutedto10nMwithddH2O.Thereactionmixturecontaining4μl5×ligation,2μlpcDNA6.2-GW/EmGFP-miR,4μl10nMdsoligo,1μl1U/μlT4DNAligaseand9μlddH2Owasplacedfor9530minatroomtemperature.TheproductswerethentransformedintoE.coliDH5αcompetentcells.Monoclonalcolonywasselected.Next,plasmidswereextractedandsequenced.Thefourplasmids(14MR0054-01~14MR0054-04)wererespectivelyrecombinedintopDONR221vectorbyInvitrogenBPcarrierrecombinationsystem.100μlofDH5αcompetentcellswastransformedby5μlrecombinationreactionsolution.Positivecloneswerescreenedandsequenced.Purposesequencewas100furtherrestructuredtopLenti6.3/V5-DESTvectorbyLPrestructuringsystem.DH5αcompetentcellsweretransformedbyrecombinationreactionsolutionagain;positivecloneswereselectedandsequenced.Ourresultsprovedthatlentiviralvectorcarryinginterferencesequencehasbeensuccessfullyachieved.Thenamesofthefourlentivirusvectorswereshownintable2.105-3- 中国科技论文在线http://www.paper.edu.cnTab.2ThelentivirusvectorsNO.ThenameoftheplasmidThenameofLentivirusvector114MR0054-01LR-2pLenti-01214MR0054-02LR-2pLenti-02314MR0054-03LR-2pLenti-03414MR0054-04LR-2pLenti-041.3Packaginglentivirus6Whenthe293Tcellsreached70%-80%confluence,cellswereplatedat1.0×10cellsper10cm110cellculturedishandculturedovernight.Themediacanberemoveddirectlybeforetransfection.9μgofPackagingMixand3μgofthelentiviralexpressionplasmidswereaddedto1.5mlOpti-MEMmediumothatwasthenpreheatedat37C.36μllipofectamine2000wasaddedto1.5mlOpti-MEMmediumandmixedgently;then,themixwaskeptatroomtemperaturefor5min.Next,wemixedplasmidsolutionandlipofectamine2000diluentandputthematroomtemperaturefor20min.Then,3mlofPlasmid115liposomecomplexwasaddedintocelldishandincubatedat37°Cin5%CO2for6h,afterwhichweremovedtheprimarymediumandreplaceditwithfreshDMEMwith10%FBS.Thesupernatantwascollectedat48hbycentrifugationat3000rpm/minfor10minandfiltratedby0.45μmmembranefilterunit.Thevirusstocksolutionwasultracentrifugedat50000gfor2h;thesupernatantwasremovedandresuspendedinopti-MEMculturemediumtodeterminethetiter.Lentiviralstocksshouldbealiquoted120andstoredat-80°C.1.4DeterminationofviraltitersTab.3Virusdilutions.StepVirusin100μl-31No.1diluent:5μlviralstock+245μLDFPmedium2×10ml-42No.2diluent:5μlNo.1diluent+245μLDFPmedium2×10ml-53No.3diluent:5μlNo.2diluent+245μLDFPmedium2×10ml-64No.4diluent:5μlNo.3diluent+245μLDFPmedium2×10ml-75No.5diluent:5μlNo.4diluent+245μLDFPmedium2×10ml-87No.6diluent:5μlNo.5diluent+245μLDFPmedium2×10mlNote:DFPmediumisDMEMcontaining20%FBSand8μg/mlPolybrene.3HEK293Tcellswereseededinto96-wellplateatadensityof8×10cellsperwell.Lentiviruswas125dilutedwithDMEMsupplementedwith2%FBSand8μg/mlPolybrene(DFPmedium).Thedetailsoftheviralstockdilutionwereshownintable3.Then,wecarefullyremovedtheculturemediumfrom96-wellplateandreplaceditwith100μldilutedvirussolution.Thecellswereincubatedinat37°Cwith-4- 中国科技论文在线http://www.paper.edu.cn5%CO2.Aftertransfectionfor24hours,GFPexpressionwasobservedunderfluorescencemicroscope,virustiterwascalculatedaftertransfectionfor96hours.LentivirusvectorsofpLenti-01~pLenti-04130weretransducedintoHEK293Tcells,whichwerescreenedunderthesameviewinfluorescentandbrightfieldsofmicroscope.1.5DeterminationofMOIvalueThebovinemusclesatellitecellsweremaintainedinDMEMsupplementedwith20%ofFBSand410%HS.Aftertheconfluence,thecellsweretrypsinizedandcounted,1×10cellswereseededin13596-wellplateswith2mlofmedium,whichwereincubatedovernight.AccordingtoMOIvalueof2,5,10,20,50,100,200,300,wedilutedlenti6.3-RNAiwithDMEMsupplementedwith2%FBSand8μg/mlpolybrene.CellswerelightlywashedoncewithPhosphateBufferedSaline(PBS)andlentivirusliquidwithdifferentMOIvalueswasaddedtothecorrespondingwellsrespectively.After72hours,theexpressionofGFPwasobserved.1401.6RNAextractionTotalRNAwasextractedfrombovinemusclesatellitecellsamplesusingTrizolbuffer,andthefollowingreversewasperformedusingaHigh-capacitycDNAReverseTranscriptionKit.1.7qRT-PCRTheqPCRreactionsystemwas17.3μlultrapurewater,2.5μl10×PCRbuffer,2μlmagnesiumions,1450.2μldNTPs,0.5μlprimers,0.5μl50×sybr,1.2μlcDNAand0.3μlofTaqDNApolymerase.ThePCRamplificationconditionswere:2minat95°C;40cyclesof95°Cfor10s,30sat60°Cand45sat70°C.TheexpressionlevelofmRNAwasdeterminedbycyclethreshold(Ct)normalizedwiththatof−ΔΔCtPPP1R11usingthe2formula.Samplestransfectedwithnegativeinterferencevectorwereusedasacontrol.1501.8DataAnalysisSYBRGreenandcDNAwereusedforQuantitativeReal-timePCR(RT-PCR).Theexpression−ΔΔCtlevelwasquantifiedvia2.AnalyticaldatawasnormalizedtothemRNAexpressionlevelofendogenouscontrolβ-actin.Primersequencesweredescribedintable4.Tab.4PrimerinformationforqRT-PCRGenePrimersequence(5"to3")Productlength(bp)PPARαF:5γ"-TCCCTCTTTGTGGCTGCTAT-3"168bpR:5"-TCGTCAGGATGGTTGTTCTG-3"PPARγF:5"-GCGACTTAGCAATATTTATAGCTGTC-3"105bpR:5"-AGGCTTGCAGCAGATTGTCT-3"RXRαF:5"-GCCTCAATGGTGTCCTCAAAG-3"120bpR:5"-AGCTGTACACCCCGTAGTGCTT-3"ApoEF:5"-AGCTGCTCAACACCCAGGTCATT-3"80bp-5- 中国科技论文在线http://www.paper.edu.cnR:5"-CTCCTTGTAGGCCTTCACCTCCTT-3"ABCA1F:5"-GGATATCCTGAAGCCAGTCCT-3"84bpR:5"-AAAGCTTTTGTGGCTTCACC-3"LPLF:5"-GACAGGATGTGGCCAAGTTT-3"61bpR:5"-TTGCCCAGGGGATAGTTAAA-3"o155Note:Annealingtemperatureforallprimersinthistableis60C.PPARα,peroxisomeproliferator-activatedreceptorα;PPARγ,peroxisomeproliferator-activatedreceptorγ;RXRα,retinoidXreceptorα;ApoE,apolipoproteinE;ABCA1,ATP-bindingcassettetransporterA1;LPL,#Lipoproteinlipase.Theprimersequencesarefrombovine.2Results1602.1Lentiviralpackaging,thetiterdeterminationresults293Tcellswerefirsttransfectedwithlentivirusvectorsfor24hrs,andthenobservedunderfluorescent(Fig.1A-D)andlight(Fig.1a-d)microscopes.SignificantGFPcouldbefoundafter-8transfectionwithvirusin100μLat2×10ml.ThevirustitersforLenti-14MR0054-01,-02,-03and-048888were3×10,2×10,2.5×10and3.5×10TU/ml,respectively.165Fig.1293TcellsweretransfectedwithplasmidscontainingspecificshRNAstargetingLXRαNote:293TcellsweremonitoredaftertransfectionwithpLenti-01,-02,-03and-04lentivirusvectorsat24hunderafluorescencemicroscope(A-D)andalightmicroscope(a-d),respectively.2.2LXRαmRNAexpression170BovinemusclesatellitecellswereinfectedwithpLenti-01,-02,-03and-04lentivirusesfor48h,andthenthesecellswerecollectedqRT-PCRanalysis.WefoundthattheLXRαmRNAexpressionlevelsweredecreasedto0.46,0.23,0.12and0.29ascomparedtonegativecontrollentivirus.Thesilencingefficienciesofthefourviruseswere54%,77%,88%and71%,respectively(Fig.2).-6- 中国科技论文在线http://www.paper.edu.cn175Fig.2KnockdownefficienciesofLXRαshRNAvectorsinbovinemusclesatellitecellsNote:BovinemusclesatellitecellswereinfectedwithpLenti-01,-02,-03and-04lentivirusfor48h,andthentheLXRαmRNAexpressionlevelsweredeterminedbyqRT-PCR.2.3LXRαrelatedgenesmRNAexpressionToexploretheeffectsofLXRαknockdownonLXRαrelatedgenes,theexpressionofPPARα,180PPARγ,ABCA1,LPL,RXRαandapoEwerealsodeterminedwithbyqRT-PCRanalysis,andtheresultsweresummarizedinfig.3.Ascomparedwithnegativecontrolgroup,themRNAexpressionlevelsofPPARα,PPARγ,ABCA1,LPLandapoEweredecreasedinbovinemusclesatellitecellswhenLXRαwasinhibited,whereasthatofRXRαwasincreased(Fig.3).LXRαplayedanegativeroleinregulating.Theresultsshowedthatup-regulationofRXRαexpressionwasinvolvedinLXRαsuppression;185therefore,theexpressionofLXRαandRXRαwerenegativelycorrelated.Fig.3EffectsofLXRαsilencingonmRNAexpressionofrelatedgenesinbovinemusclesatellitecellsNote:Thesedatarepresentthemeans±S.D.*representsP<0.05.TheSameasfollows.ovinemusclesatellitecellswereinfectedwithpLenti-01,-02,-03and-04lentivirusfor48h,andthenthemRNAexpressionlevelsof190(A)PPARα,(B)PPARγ,(C)ABCA1,(D)LPL,(E)ApoEand(F)RXRαweredeterminedbyqRT-PCR.-7- 中国科技论文在线http://www.paper.edu.cnTheinterferenceefficiencyofpLenti-03vectorwas88%,whichwasthebestamongallthevectors.TherelativeexpressionofthesixgenesregulatedbypLenti-03wasshowninfig.4.Comparedwiththenegativecontrolgroup,exceptRXRα,theexpressionleveloftheotherfivegenesdecreasedlessthanone.TherelatedgenesdecreasedbyLXRαwereintheorderofPPARγ>PPARα>LPL>195apoE>ABCA1.TherelativeexpressionofRXRαgenewashigherthanthenegativecontrolandwasup-regulated.Hence,thesilencingeffectsofbovineLXRαgeneonPPARα,PPARγ,ABCA1,LPL,RXRαandapoEweresignificant.Fig.4TheeffectsofpLenti-03infectiononbovinemusclesatellitecells200Note:RelativemRNAexpressionofPPARα,PPARγ,ABCA1,LPL,ApoEandRXRαgenes.3DiscussionBovinemusclesatellitecellswereusedinourexperiment.thepositiverateofGFPwasveryhighwhencellwasinfectedbyadenoviruswithMOIvalue3000,butthepositiveofcellularfluorescenceintensitywasverylow.Thereasonforthiswasthatadenoviruscannotbeappliedtoinfectbovine205musclesatellitecells.However,thepositiverateofthecellswasveryhighwhenlentivirusinfectingbovinemusclesatellitecellswithMOI300.Althoughthepositiveratewaslowandcellularfluorescenceintensitywasweak,whichalsohaveanobviouseffectonthecellularshape.Lentiviralvectorwasdevelopedbasisonthegenetherapyofthehumanimmunodeficiencyvirusandhavethe[18,19]highefficiencyforinfectionandstablesilencingtargetgene,therefore,itwasusedwidely.210Lentivirus,asgenetransfervector,wascharacterizedbyhightransfectionefficiencyinnon-dividingcellsanddividingcells,washightiter,goodstabilityintargetcellsandlittleimmunoreactivity,andhas[20]beenwidelyusedintransformingintovectorofgeneticengineering.Comparedtoanothercarrierlentiviralvector,ithasitsuniqueadvantages:forsomedifficulttotransfectcellswhichhasahighinfectionrateforprimarycells,stemcellsandundifferentiatedcells,andthegeneticrecombination215couldnotoccurandthevectorcouldbestablyexpressed.Inthisstudy,wefoundthatLXRαgenesweredown-regulatedbyeighty-eightpercentafterlentivirusinfectingbovinemusclesatellitecellsfor48h,theexpressionofABCA1andApoEwerealsodecreased.Meanwhile,LXRαcouldincreasetheexpressionofATP-bindingcassettetransporterprotein-8- 中国科技论文在线http://www.paper.edu.cnandpromotecholesterolefflux.Additionally,afteractivatingLXRαinmacrophages,theexpressionof220ABCA1,ABCG1andApoEalsoincreased.LXRandPPARpathwaycouplingincreasedexpressionof[21]ABCA1andregulatedlipidintakeandreversedtransport.LXRαgenesmightdecreasetheexpressionofApoE.Comparedwiththecontrolgroup,theexpressionofPPARγwasdecreasedinbovinemusclesatellitecells.WhentheexpressionofLXRαgeneisincreased,moreheterodimerscouldbeformedbyPPARγandRXR,inhibitingPPARγandRXRformingheterodimersanddecreasingtheexpressionof[22]225PPARγ.Therefore,whenLXRαwasdown-regulated,LXRαandPPARγcouldformacompetitivemechanism,whichcauseddecreasedexpressionofPPARγ.However,themechanismofup-regulatedexpressionofRXRαhasnotbeenidentifiedandthefurtherstudyshouldberemainedandinvestigated.ItisofgreatsignificancetostudyintracellularlipidmetabolicthroughinterferingLXRαgene.LentivirusinterferingvectorlaysthefoundationforfurtherstudyingLXRαgenefunctioninlipid230metabolisminbovinemusclesatellitecells.4ConclusionlentiviralvectorcarryingtheshRNAtargetingLXRαgenewassuccessfullyconstructed,andthelentiviralvectorcanbeefficientlyexpressedinbovinemusclesatellitecells.WealsoexploredtheeffectsofLXRαgenesilencingonthemetabolicassociatedgenes.Thisstudyprovidesnewinsights235intotheregulationofbovineLXRαincholesterolmetabolism.AcknowledgementsThisworkwassupportedbytheresearchfundforthedoctoralofhighereducationofChina(20130202120008),theChinapostdoctoralsciencefoundation(2015M570811)andthefundamentalresearchfundsforthecentraluniversitiesofChina(GK201502008).240References[1]ThomasPB,LauraAS,WangYJ,etal.Nuclearreceptorsandtheirselectivepharmacologicmodulators[J].PharmacoRev,2013,65:710-778.[2]SavkurRS,Burris,TP.ThecoactivatorLXXLLnuclearreceptorrecognitionmotif[J].TheJournalof245PeptideResearch,2004,63:207-212.[3]ZelcerN,TontonozP.LiverXreceptorsasintegratorsofmetabolicandinflammatorysignaling[J].JClinInvest,2006,116:607-614.[4]LundEG,MenkeJG,SparrowCP.LiverXreceptoragonistsaspotentialtherapeuticagentsfordyslipidemiaandatherosclerosis[J].ArteriosclerThrombVascBiol,2003,23:1169-1177.250[5]OstlundJr.RE.Aminimalmodelforhumanwholebodycholesterolmetabolism[J].AmJPhysiol,1993,265:E513-E520.[6]ZelcerN,TontonozP.LiverXreceptorsasintegratorsofmetabolicandinflammatorysignaling[J].JClinInvest,2006,16(3):607-614.[7]WillyPJ,UmesonoK,OngES,etal.LXR,anuclearreceptorthatdefinesadistinctretinoidresponse255pathway[J].GenesDev,1995,9:1033-1045.[8]VinodM,ChennamsettyI,ColinS,etal.miR-206controlsLXRαexpressionandpromotesLXR-mediatedcholesteroleffluxinmacrophages[J].Biochim.Biophys.Acta,2014,1841(6):827-835.[9]IshimotoK,TachibanaK,SumitomoM,etal.Identificationofhumanlow-densitylipoproteinreceptorasanoveltargetgeneregulatedbyliverXreceptoralpha[J].FebsLett,2006,580:4929-4933.260[10]SoumianS,AlbrechtC,DaviesAH,etal.ABCA1andatherosclerosis[J].VascMed,2005,10:109-119.[11]ChawlaA,BoisvertWA,LeeCH,etal.APPARgamma-LXR-ABCA1pathwayinmacrophagesisinvolvedincholesteroleffluxandathero-genesis[J].MolCell,2001,7(1):161-171.[12]WongJ,QuinnCM,GelissenIC,etal.TheeffectofstatinsonABCA1andABCG1expressioninhumanmacrophagesisinfluencedbycellularcholesterollevelsandextentofdifferentiation[J].Atherosclerosis,2008,265196:180-189.[13]HuYW,MaX,HuangJL,etal.DihydrocapsaicinAttenuatesPlaqueFormationthroughaPPARγ/LXRαPathwayinapoE-/-MiceFedaHigh-Fat/High-CholesterolDiet[J].PLoSOne,2013,8(6):e66876.-9- 中国科技论文在线http://www.paper.edu.cn[14]ZhangY,RepaJY,GauthierK,etal.RegulationofLipoproteinLipasebytheOxysterolReceptors,LXRαandLXRβ[J].JBiolChem,2001,276:43018-43024.270[15]HannonGJ.RNAinterference[J].Nature,2002,418(6894):244-251.[16]SongE,LeeSK,WangJ,etal.RNAinterferencetargetingFasprotectsmicefromfulminanthepatitis[J].NatMed,2003,9:347-351.[16]SongE,LeeSK,WangJ,etal.RNAinterferencetargetingFasprotectsmicefromfulminanthepatitis[J].NatMed,2003,9:347-351.275[17]JacqueJM,TriquesK,StevensonM.ModulationofHIV-1replicationbyRNAinterference[J].Nature,2002,418:435-438.[18]WangS,ZengX,LiuY,etal.ConstructionandcharacterizationofaPDCD5recombinantlentivirusvectoranditsexpressionintumorcells[J].OncolRep,2012,28(1):91-98.[19]ZhaoB,YangC,YangS,etal.Constructionofconditionallentivirus-mediatedshRNAvectortargetingthe280humanMirkgeneandidentificationofRNAiefficiencyinrhabdomyosarcomaRDcells[J].IntJOncol,2013,43(4):1253-1259.[20]KohDM,BrownG,CollinsDJ.Nanoparticlesinrectalcancerimaging[J].CancerBiomark,2009,5(2):89-98.[21]TobinKA,SteinegerHH,AlbertiS,etal.Cross-talkbetweenfattyacidandcholesterolmetabolism285mediatedbyliverXreceptor-alpha[J].MolEndocrinol,2000,14(5):741-752.[22]YoshikawaT,IdeT,ShimanoH,etal.Cross-talkbetweenperoxisomeproliferator-activatedreceptor(PPARα)andliverXreceptor(LXR)innutritionalregulationoffattyacidmetabolismIPPARssuppresssterolregulatoryelementbindingprotein-1cpromoterthroughinhibitionofLXRsignaling[J].MolecularEndocrinology,2003,17(7):1240-1254.290牛LXRα基因mRNA重组慢病毒载体的构建及其对牛肌肉卫星细胞的干扰效果刘永峰，赵璐，库婷，杨兴斌295（食品工程与营养科学学院，陕西师范大学，西安，710062）摘要：肝X受体α(LXRα)基因是核激素受体家族的成员，它可以调节参与胆固醇代谢基因的转录。近期研究发现LXRα被认为是一个调节巨噬细胞内胆固醇平衡的关键因子，从而调节多个基因参与胆固醇运输，如ATP结合蛋白(ABCs)、ABCA1、ABCG1、载脂蛋白E(ApoE)、脂蛋白脂肪酶(LPL)等功能基因。本研究中，以牛LXRα基因为目标基因，设计300了4对shRNA，使用BP和LP重组系统将其构建到慢病毒载体上，在293T细胞中转染获得了相应滴度的载体。Lenti-14MR0054-01、-02、-03和-04四个慢病毒分别以3×108TU/mL、2×108TU/mL、2.5×108TU/mL和3.5×108TU/mL的滴度侵染牛肌肉卫星细胞，定量筛选了最佳载体为pLenti-03，干扰效率达到88%。此外，发现pLenti-03侵染48h后，牛RXRα基因的mRNA表达为上调，同时牛PPARα、PPARγ、ABCA1、LPL和ApoE基因的305mRNA表达为下调。因此，本研究成功构建了高效抑制牛LXRα表达的慢病毒载体，为在牛肌肉卫星细胞中降低LXRα的作用提供了理论基础。关键词：牛LXRα基因；慢病毒；载体构建；基因干扰中图分类号：S858.23-10-'