简介：Genestellcellswhattodo,forexample,whentorepairDNAmistakesorwhentodie,andcanbeturnedonorofflikealightswitch.Knowingwhichgenesareswitchedon,orexpressed,isimportantforthetreatmentandmonitoringofdisease.Now,forthefirsttime,Researchersinthelaboratoryof

简介：Objective:ToconstructsurvivinshRNAexpressionvectorcartingenhancedgreenfluorescentproteingene,transfectitintoGBC-SDHcellsviaelectroporation,andgetGBC-SDcellswhicharestableexpressingsurvivinshRNA.Methods:ThesiRNAsequencetargetingsurvivinmRNAwassynthesizedandclonedintopEGFP-H1.TheconstructedplasmidandpEGFP-H1weretransfectedintoGBC-SDcellsrespectivelyvialiposome,andthetransfectingeffectwasdetectedwithFlowCytometry.ThenthetransfectedcellswereselectedwithG418.Results:Therecombinantplasmidwassuccessfullyconstructed,namedpEGFP-survivin.ThegenetransfectionefficienciesinpEGFP-H1-transfectedgroupandpEGFP-survivin-transfectedgroupwerethe80.29%±2.71%and83.85%±2.34%(P>0.05),whichwassuccessfultogetthecellsthatarestableexpressingshRNA,namedGBC-SD/EGFPandGBC-SD/survivin.Conclusion:SurvivinshRNAexpressionvectorwasconstructedsuccessfullyandgotGBC-SDcellswhicharestableexpressionshRNA.

简介：Objective:Toachieveanoptimizedmethodforsolubleexpressionofhumancarboxylesterase1(hCE-1)inescherichiacoilandpurificationbyNi2+-NTAagaroseaffinitychromatography,togetimprovedproteinyieldandpurityforfurtherdevelopmentofhepatocellularcarcinoma(HCC)diagnosisELISAkits.Methods:ThebestantigenepitopesofhCE1werepredictedbycomparingsecondarystructure,flexibleregions,hydrophilicity,antigenicindexsurfaceprobabilityofresidues.Afterwards,pET-42a(+)withaHis-tagandaGST-tagwasappliedtoformrecombinantplasmidpET-42a(+)/hCE1,whichfacilitatedpurificationwhenusingNi2+-NTAagaroseaffinitychromatography.ProteinqualitywasmeasuredbySDS-PAGEandBCAproteinassay.Western-blotidentificationwasalsoperformedtoensurethecorrectexpressionofhCE1protein.Results:Theresiduesfrom500to567nearC-terminalofhCE1proteinwereconsideredthebestepitopeswhichexhibitedhighhydrophilicityandhighsurfaceprobabilityandrelativelyflexiblesecondarystructureandlowhomologycomparedwithhCE2andhCE3.His-hCE1500-567fusionproteinwasachievedbyIPTG-inductedexpressionwithanexpectedmassof42kDa.Afterpurification,thefinalproductwasspeciallyidentified,whichreachedover95%purityandmorethan10mg/Lofmicrobialculture.InWesternblot,thepurifiedfusionproteinwasrecognizedbyanti-hCE1monoclonalantibody,alongwithprevioussequencingvalidation,whichdemonstratedthecorrectpreparationofsolublehCE1protein.Conclusion:ThisisanefficaciousandaffordablestrategytogeneratefusionhCE1ofhighqualityinEcoli,whichfacilitatespreparationofhCE1monoclonalantibodyandfurtherHCCdiagnosisresearch.

简介：Inthispaper,theexperimentalandnumericalstudiesofsteadyturbulentflowfieldofbileafletmechanicalvalvewithcamberedprofiearepresented.WithLaserDopplerAnemometry,theauthormeasuredtheflowofheartvalveinvitro.Theworkingfluidwaswater,theflowrateswere15,18,21,28and38l/minrespectively.ThecorrespondingReynoldsnumberswere11789,14147,16505,22006and29866.

简介：LargeconductanceCa~(2+)-activatedK~+(BK_(Ca))channelexhibitsaphenotype-dependentexpressiononvascularsmoothmusclecells(VSMCs),whichpreferstocontractilephenotype.Meanwhile,shearstressdefinitelyinfluencesVSMCsproliferationandcontraction.Thereby,ahypothesiswasraised,wouldshearstresschangetheBK_(Ca)expressionandcorrelatewithVSMCphenotype?Inordertoinvestigateit,VSMCswereexposedtoshearstressinaparallel-plateflowchamberwith12dynes/cm~2for12h.Subsequently,theeffectofshearstressonVSMCproliferation,BK_(Ca)channelexpressionandcontractilephenotypemarker,α-smoothmusclecellactin(α-SMA)andsmoothmusclemyosinheavychain(SMMHC),wasdeterminedbyimmunofluorescencemicroscopy,flowcytometeryaswellasreversetranscriptionpolymerasechainreaction(RT-PCR),respectively.DatashowthatshearstressenhancedtheexpressionofBK_(Ca)channelwhileinhibitingVSMCproliferation.Paralleledtothosephenomena,theexpressionofbothα-SMAandSM-MHCweredecreasedsignificantly.TheseresultsdemonstratedthatupregulationofBK_(Ca)channelwasirrelevanttothemaintenanceVSMCofcontractilephenotypeundershearstress.ThisfindingprovidesanewinsightintounderstandingthecorrelationofBK_(Ca)channelandVSMCphenotype.

简介：IntroductionAsamemberofthebonemorphogeneticprotein(BMP)family,BMP-2playsimportantrolesnotonlyinboneregenerationandbonerepairbutalsoincellproliferation,apoptosis,differentiationandmorphogenesis.TheBMP-2remarkableabilitytostimulatenewbonegrowthresultsinthedevelopmentofanoveltherapystrategyforbonemassdefectduetoaccidentsordiseases.BecausetheBMP-2itself,inconjunctionwithasuitablematrix,issufficienttostimulategenesisofnewbone,thegeneticallyengineeredBMP-2hasgoodappliedprospects.

简介：Objective:Thispaperaimsatmeasurementenhancedeffectofoxidizedlipoprotien(a)[oxLp(a)]onpermeabilityofmonolayerendothelialcellsandrelationshipwithreactiveoxygenspecies(ROS)generationanddesmogleins(DSGs)expression.MethodsandResults:Transendothelialpermeabilitywasassayedbytranswellandreactiveoxygenspecies(ROS)wasdeterminedbyDCFH-DAstaining.RT-PCRwascarriedouttodetermineDSGlandDSC2expressioninmRNA,respectively.TransendothelialpermeabilitywasenhancedbyoxLP(a)doseandtimedependently.Themostmarkedeffectappearedataconcentrationof100mg/L,Transendothelialpermeabilityreachedthemaximumvalueafter2hofFITC-dextranaddition,andthengraduallydecreasedafter4h.oxLp(a)inducesthegenerationofcellularreactiveoxygenspecies(ROS),andthiseffectcouldbeinhibitedbysuperoxidedismutase(SOD).IncubationofHUVECswithoxLp(a)resultedinadoseandtime-dependentdown-regulationofDSGlandDSC2expressionattranscriptionallevel.Conclusion:PermeabilityofmonolayerendothelialcellswasenhancedbyoxLp(a)whichisrelatedtoup-regulatingROSformationanddown-regulatingdesmogleinsexpression.

简介：Objective:ThispaperistoexploreamethodoftransferringhumanSDF-1anditsmutantSDF-1/54intrakinegeneintoCOS-7cellsfordeterminingtheirexpressionandsubcelluarlocalizationofthefusionprotein.Thiscouldofferfeasibilityforinhibitingthemetastasisofmalignanttumorsbyphonotypicknockoutforblockingfunctionalexpressionofreceptoronthecell-surface.Methods:AmplifythetargetgenewithPCRfromtheconstructedplasimidSDF-WT-Gly×4-Dec/PET-30a(+)withaC-terminalretentionsignalfragmentKDEL.AfterthepcDNA3.1/SDF-1/KDEL,pcDNA3.1/SDF-1/54/KDEL,pEGFP/SDF-1/KDELandpEGFP/SDF-1/54/KDELeukaryoticexpressionvectorswereconstructedandtheDNAsequencewasaccurate,theyweretransferredintoCOS-7cellswithliposome.Theexogenousexpressionswereobserved,fusionproteinSDF-1/HisandSDF-1/54/HiswereconfirmedbyWesternblot,andtheSDF-1/EGFPandSDF-1/54/EGFPweredeterminedbyLaserScanningConfocalMicroscopy.Results:Fourexpressionvectorswereconstructedsuccessfully,thefusionproteinSDF-1/KDEL/HisandSDF-1/54KDEL/HisexpressedinCOS-7cells.SubcelluarlocalizationanalysisshowedthatSDF-1/KDEL/EGFPandSDF-1/54/KDEL/EGFPwerelocatedmainlyinendoplasmicreticulum.Conclusion:FourexpressionvectorspcDNA3.1/SDF-1/KDEL,pcDNA3.1/SDF-1/54/KDEL,pEGFP/SDF-1/KDELandpEGFP/SDF-1/54/KDELwereconstructedsuccessfully,whichcouldexpressineukaryoticcellandlocatemainlyintheendoplasmicreticulum.

简介：Curcumin,adietaryphytochemical,exhibitsmultifunctionalnaturalproductwithregulatoryeffectsoninflammation.However,thepoorbioavailabilitylimitsitsclinicalapplications.Thus,wedesignedandsynthesizedanovelmonocarbonylanalogueofcurcuminB7andtheirinhibitionagainstmonocytechemotacticprotein-1（MCP-1）andinterleukin-8（IL-8）releasewasevaluatedinH2O2-stimulatedhumanvascularendothelialcells（ECs）inadose-responsivemanner,whileexhibitingnocytotoxicityinECs.Takentogether,theseinsightsonthenovelcompoundB7mayserveaspotentialagentsforthetreatmentofatherosclerosis.