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：Ourobjectiveistosolvethelactosemalabsorptionandintoleranceofhumanbeingsbycombiningmlcro-ecologypathwithgeneticengineeringtechnique.PlasmidpMG36ewasusedtocloneandexpressaβ-galactosidasegenefromL.delbrueckiibulgaricusstrain1.1480intheLactococcuslactissubsp,cremorisMG1363andLactococcuslactissubsp.lactisIL1403.TherecombinantplasmidwaspreservedandproliferatedinEscherichiacoli(E.coli)JM109,andtransformedintoMG1363and1L1403byelectroporation.Theproteinexpressionwasstudied.(1)Thebifidobacteriumculturemedium(BBL)wassuitableforthegrowthofthestrain1.1480.(2)With13aminoacidsattheN-terminusfromthevector,β-galactosidasefusionprotein(whichretainedtheenzymeactivity)couldbesuccessfullyexpressedinE.coliJM109,MG1363andIL1403,buttheexpressionquantitywaslargerintheformerthaninthelattertwo.(3)TheSDsequencedesignedcouldbesuccessfullyrecognizedbyboththeE.coliandtheLactococcuslactis,buttheexpressionlevelofthenon-fusionβ-galac-tosidaseproteinwaslowerthanthatofthefusionproteininthesamehost.Theβ-galactosidasegeneticallyengineeredE.coliJM109isausefultooltoproducethisenzymeinvitro.Thesignalpeptideoftheusp45proteinfromtheLactococcuslactiscanbeaddedbeforethepromotersequencetopromoteβ-galactosidasesecretionfromLactococcuslactis.Thepotentialapplicationoftheβ-galactosidasegeneticallyengineeredMG1363andIL1403tocurethelactosemalabsorptionandlactoseintoleranceinbothhealthfoodandmedicineispromising。

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简介：Theeffectsofvariouscartilageextracellularmatrixontheconstructionofrabbitgrowthplatecartilagetissueinvitrowerestudied.Theresultsshowthatcollagen,proteoglycanandhyaluronicacidcanpromotethegrowthofculturedchondrocytesbuttheeffectsofvariouscartilageextracellularmatrix(ECM)onchondrocytedifferentiationaredifferent.Collagencanpromotethehypertrophyofchondrocyteswhileproteoglycanandhyaluronicacidinhibitthetransitionofmaturechondrocytesintohypertrophiedchondrocytes.

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简介：Objective:Toprovideasoundcellsourceforfurtherex-vivogenetherapyforchronicpain,weattempttodevelopanimmortalizedratastrocytecelllinethatexpressesenkephalinregulatedbydoxycycline.Methods:Retrovirusinfectionmethodwasemployedtodevelopanimmortalizedratastrocytecelllinethatcouldexpressenkephalinregulatedbydoxycycline.ThehPPEgeneexpressionlevelofimmoralizedastroytecells(IAC)/hPPEwasdetectedbyRT-PCR,indirectimmunofluorescencestainingandradioimmunoassay.Results:IACcarryingTet-onsystemtransfectedwithpreproenkephalingenecouldsecreteenkephalinthatwasregulatedbydoxycyclineinadose-dependentmannerandhPPEgeneactivationcouldberepeatedinon-off-oncyclesthroughadministrationorremovalofdoxycycline.Conclusion:Animmortalizedratastrocytecelllinethatsecreteenkephalinunderthecontrolofdoxycyclineisestablishedsuccessfully,whichprovidesaresearchbasisfortransgeniccelltransplantationforanalgesia.

简介：INTRODUCTIONThecommonconstructionmethodoftissueengineeredarticularcartilageistoisolateandculturechondrocytes,transplantthemintothedegradablescaffoldmaterials,thentorepairthedefectofarticularcartilage.Wedesignedthisexperimenttoinvestigatethefeasibilityoftissueengineeredarticularcartilageconstructedbythetechniqueofcentrifugetubeculturewithoutscaffoldmaterials.

简介：Analysisofthefrequencyofantigen-specificcytotoxicTlymphocytes(CTLs)exvivoislargelydependentontheuseofMHC/peptidetetramers.However,thelatterreagentshavenotbeenwidelyavailable,mostlikelybecauseoftheircostlyandtime-consumingproduction.InthisreportweutilizedaneconomicstrategytoconstructHLA/peptidetetramerswithrecombinantpeptide-linkedβ2microglobulin(β2m).TheHLA-A2-restricted,melanomaantigenMARTl-derivedpeptideMARTI27-35(AAGIGILTV)wasfusedtotheNterminusofhumanβ2mthrougha15-aminoacid(aa)-longlinkerbeforebeingrefoldedwiththerecombinantbiotinylatedHLA-A2heavychainectodomain.Theresulted2-component(2C)monomerwasthentetramerizedwithphycoerythin-labeledstreptavidin.Theexperimentalresultshowedthatthe2CHLA-A2/MARTI27-35monomerwasshowntobindtotheHLAclassⅠcomplex-specificmonoclonalantibodyW6/32andtheHLA-A2/MARTI27-35complex-specificsinglechainantibodyfragment(scFv)8.3,suggestingthecorrectnessofitsspecificity.Furthermore,the2CHLA-A2/MARTI27-35tetramerdetectedaspecificCD8^+TcellpopulationinHLA-A2-restrictedmelanomainfiltratinglymphocytesastheconventional3CHLA-A2/MARTI27-35tetramer.Theyieldof2CHLA-A2/MARTI27-35monomerwas2.5timesmorethanthatoftheconventional3Cmonomer.Takentogether,thesedataindicatethattheHLA-A2/MARTI27-35tetramercanbegeneratedconvenientlythroughtheuseofMARTI27-35peptide-β2mfusionproteins,whichcanfacilitatethemonitoringofHLA-A2-restricted,MARTl-specificCTLresponsesinpatientswithmelanoma.

简介：Theresearchgroupwassuccessfulingeneratingavasculargraftwithbiomimeticcircumferentialtensilestrengthandexpressionofsmoothmusclecellspecificgenesoverstaticculture.

简介：AIMTo估计角膜的矩阵作为脚手架过去常重建的非细胞组成的驼鸟张力亢进的盐溶液与一个消化方法相结合的损坏cornea.METHODSA习惯于decellularize驼鸟角膜。非细胞组成的角膜的矩阵的微观结构被传播电子显微镜学(TEM)和hematoxylin和曙红观察(H&；E)染色。机械性质被一个电流计和一台紧张机器检测。非细胞组成的角膜的矩阵也被移植进一个兔子角膜和cytokeratin3被用来检查有免疫力的phenotype.RESULTSThe微观结构和驼鸟角膜的机械性质很好在decellularization过程以后被保存。在vitro，甲基thiazolyltetrazolium结果表明非细胞组成的驼鸟角膜(AOC)的摘录没在增长上有禁止的效果角膜上皮或endothelial房间或在keratocytes上。兔子当角膜的混浊和接枝溶解发生在非细胞组成的猪的角膜(APC)时，薄片状的keratoplasty证明移植AOC进主人角膜透明、完全合并移植。重建的角膜的显型类似于有3首先在表面的上皮的房间layer.CONCLUSIONWe使用了的cytokeratin的高表情的一个正常兔子角膜重建的脚手架损坏了角膜的AOC。与猪的角膜相比，驼鸟角膜的解剖结构人的角膜接近那些。根据原则，那结构决定功能，薄片状的keratoplasty也证实了的异种皮移植AOC移植与APC接枝的相比产生了优异结果。

简介：Thisresearchusesalginateandhyaluronicacidasthemaincomponenttopreparesupport,thenexploresthepossibilitiesasatissueengineeringscaffold.Firstly,prepareHAwithvariousaveragemolecularweightandalginatewithdifferentviscosity,mixthemupatacertainproportionandmakeitintoaAlgCa2+-HAcompositescaffoldwithafilm-formingmethod.Thisarticlediscussesthefeasibilityofthisscaffoldusedintissueengineeringfieldaccordingtotheconsequenceofmoisturecontenttesting,mechanicalanalysis,andscanningelectronmicroscopyanalysis.ThestructureandpropertiesofAlgCa2+-HAcompositescaffoldarecloselyrelatedtosomefactorssuchasaveragemolecularweightofhyaluronicacid,hyaluronicacidconcentration,alginateviscosity,cross-linkingagentsandprocessingtechnology.TheAlgCa2+-HAcompositematerial,whichisatdifferentproportionsandaddingdifferentcross-linkingagent,hassomecertaincharacteristics:moisturecontentrangingfrom50%to95%,tensilestrengthbetween2.69N/mm2and4.299N/mm2,andelongationatbreakisabout58%to160%.ThepreparedAlgCa2+-HAcompositescaffoldscanbeusedastissueengineeringscaffoldsresultingfromitshighmoisturecontent,goodmechanicalpropertiesandidealporestructure.更多还原

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简介：ObjectiveToconstructaprokaryoticexpressionvectorbearingfusiongeneNT4-ADNF-9forfuturestudiesongenetictherapiesforsensorineuraldeafness.MethodsDoublestrandADNF-9cDNAwassynthesizedusingasymmetricalprimer/templatesandligatedtothe3'terminalofsignalandleaderpeptidesofneurotrophin4(NT4).ThefusiongeneNT4-ADNF-9,wassubclonedintoprokaryoticexpressionvectorpBV220,andnamedpBV220/NT4-ADNF-9.DNAsequenceofthefusiongenewasanalyzed.ThefusionproteinwasisolatedbySDS-PAGEanditsbioactivitywasevaluatedusingprimarycultureofday8chickenembryonicDRGcells.ResultsThecorrectsequenceoffusiongeneNT4-ADNF-9wassuccessfullysubclonedintothepBV220vector.TheexpressedADNF-9proteinshoweditseffectsinpromotingcellsurvivalandneuritegrowth.ConclusionProkaryoticexpressionvectorpBV220/NT4-ADNF-9wasconstructedsuccessfullyandtheexpressedfusionproteindemonstratedsatisfactorybioactivity.

简介：INTRODUCTIONApproximately400millionpersonsworldwidesufferfrombladderdisease.Individualswithend-stagebladderdiseaseoftenrequirebladderreplacementorrepair.Severalbladdersubstituteshavebeenattemptedwithbothorganicmaterialsandsynthetics.

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

简介：Inthepresentstudy,weconstructedalentivirus,FIV-CMV-GFP-miR-7-3,containingthemicroRNA-7-3geneandthegreenfluorescentproteingene,andusedittotransfecthumangliomaU251cells.Fluorescencemicroscopyshowedthat80%ofU251cellsexpressedgreenfluorescence.Real-timereversetranscriptionPCRshowedthatmicroRNA-7-3RNAexpressioninU251cellswassignificantlyincreased.ProliferationwasslowedintransfectedU251cells,andmostcellswereintheG1phaseofthecellcycle.Inaddition,theexpressionoftheserine/threonineproteinkinase2wasdecreased.ResultssuggestedthattransfectionwithalentiviruscarryingmicroRNA-7-3caneffectivelysuppressepidermalgrowthfactorreceptorpathwayactivityinU251cells,arrestcellcycletransitionfromG1phasetoSphaseandinhibitgliomacellgrowth.

简介：Prematureventricularcontraction(PVC)isthemostfrequentarrhythmiaencounteredinclinicalpractice.PVCmayoccurinhealthsubjects,whichisnotimminentlylife-threateningbutmayrequiretherapiestopreventfurtherproblems.So,thetimelyPVCrecognitionbecomesveryimportantfortheanalysisofelectrocardiogram(ECG),especiallyfortheremoteECGmonitoringusingmobilephones.Inthispaper,aconstructionmethodofpersonalizedECGtemplateandaPVCrecognitionmethodbasedontemplatematchingwerestudied.Firstly,weselected43ECGrecordingsfromtheMIT-BIHarrhythmiadatabase.Allrecordingsweredividedintotwodatasets(DS1fortrainingandDS2fortesting)andeachdatasetapproximatelycontainedthesameproportionofPVCbeats.Subsequently,foreachrecording(30min)inDS1,thefirst5minrecordingswereusedtoconstructthepersonalizedECGtemplateandthelast25minrecordingswereusedfortheR-wavepeaksdetectionandPVCrecognition,wherethetemplatematchingmethodwereused.ThevalidityoftheproposedmethodswastestedusingDS2.Theresultsshowedthat:1)highbeatdetectionaccuracywasachievedforbothPVCbeatsandnon-PVCbeats;2)thesensitivityandspecificityofPVCrecognitionwere99.11%and99.96%forthefirst5minrecordingsrespectively,99.17%and99.43%forthelast25minrecordingsrespectively.Alltheproposedmethodscanbereal-timeperformed,whichshowapromisingprospectfortheapplicationofECGmobilephones.

简介：AIM:Toconstructanon-resistantandattenuatedSalmonellatyphimurium(S.typhimurium)strainwhichexpressesconservativeregionofadhesionABofHelicobacterpylori(Hpylon)andevaluateitsimmunogenicity.METHODS:TheABgeneamplifiedbyPCRwasinsertedintotheexpressionvectorpYA248containingasdgeneandthroughtwotransformationsintroducedintothedeltaCya1deltaCrp1deltaAsdattenuatedSalmonellatyphimuriumstrain,constructingbalancedlethalattenuatedSalmonellatyphimuriumstrainsX4072(pYA248-AB).BridgedELISAmethodwasusedtomeasuretheexpressionofABantigeninsonicateandculturesupematant.AccordingtothemethoddescribedbyMeacock,stabilityoftherecombinantwasevaluated.Semi-lethalcapacitytestwasusedtoevaluatethesafetyofrecombinant.Theimmunogenicityofrecombinantwasevaluatedwithanimalexperiments.RESULTS:TheattenuatedS.typhimuriumX4072(pYA248-AB)whichexpressesABwassuccessfullyconstructed.Furthermore,bridgedELISAassayshowedthatthecontentofABinrecombinantX4072(pYA248-AB)culturesupematantwashigherthanthatwasinthalluslyticliquor.AndafterrecombinantX4072(pYA248-AB)wasculturedfor100generationswithoutselectionpressure,bheentirerecombinantbacteriaselectedrandomlycouldgrow,andtheABantigenwasdefectedpositivebyELISA.ThegrowthcurveoftherecombinantbacteriashowedthatthegrowthstatesofX4072(pYA248)andX4072(pYA248-AB)werebasicallyconsistent.ThesurvivalrateofC57BL/6wasstill100%,at30daftermicetakingX4072(pYA248-AB)1.0×l0^10cfuorally.OralimmunizationofmicewithX4072(pYA248-AB)inducedaspecificimmuneresponse.CONCLUSION:Invitrorecombinantplasmidappearstobestableandexperimentsonanimalsshowedthattherecombinantstrainsweresafeandimmunogenicinvitro,whichprovidinganewliveoralvaccinecandidateforprotectionandcareofHpyloriinfection.

简介：Objective:ToprovideahighlyefficientadenoviralvectorAd-CMV-hTGFβ1forthestudyofgenetherapyforreversionoftheintervertebraldiscdegeneration.Methods:Anewlydevelopedrecombinantadenoviralvectorconstructionsystemwasusedinthestudy.ThecDNAofhTGFβ1wasfirstsubclonedintoashuttleplasmidpShuttle-CMV.TheresultantplasmidwaslinearizedbydigestingwithrestrictionendonucleasePmeI,andsubsequentlytransformedintoE.coll.BJ5183cellswithanadenoviralbackboneplasmidpAdEasy-1.Recombinantswereselectedbykanamycinresistanceandconfirmedbyrestrictionendonucleaseanalysis.Finally,therecombinantplasmidlinearizedbyPmeIwastransfectedinto293cells.Recombinantadenovirusesweregeneratedwithin2weeks.Results:TherecombinantadenoviralplasmidswerecutbyBamHIandPacIrespectively,andthediagnosticfragmentsappearedin0.8%agaroseelectrophoresis.Theinfected293cellsshowedevidentcytopathlceffect(CPE).TheproductionsofPCRconfirmedthepresenceofrecombinantadenovirus.TheexpressionofhTGFβ1wasverifiedbyimmunohistochemicalstaining.Conclusions:ThesuccessfulgenerationoftheadenoviralvectorAd-CMV-hTGFβ1andtheconfirmationoftheinterestgeneexpressionmakeitpossiblefortheexperimentalstudyofthereversionoftheintervertebraldiscdegenerationbygenetherapy.