微环载体介导肝再生增强因子的肝纤维化的治疗

1、微环载体介导肝再生增强因子的肝纤维化的治疗researcharticlesaugme nterofliverrege nerati ongen etherapyusi nganovelminicirclednavectoralleviateslive rfibrosisinrats1,221,21,223xinwu,guangzeliu,maomu,yuti ngpe ngxiumeili 丄 iside ng,221,*andxia ngpingkon g2?*zhe nweizha ngmeijua nchens on gyou,instituteoflifescie ncea ndbio

2、 pharmaceuticsshenyangpharmaceuticalu niversit y,she ny ang,china;2keylaboratoryofliverdiseasezcentreoflnfectiousdiseases,458thhospitalofpla,guangzhou,china;3thefifthaf ?liatedhospital,s un yat-se nun iversity,zhuhai,chi na.1liver?brosisresultsi ncirrhosis,liverca ncera ndliverfailur 巳 whichisamajor

3、causeof mortalityworldwide.ge netherapyisarelatively newparadigminmedicinewithen ormoustherapeuticpote ntial.thedevelopme ntofa nef?cie ntan dsafedeliverysyst emisesse ntialforcli nicalge netherapy.lnthepresentstudyweevaluatedaugmenter ofliverrege neratio n/growthfactorervllike(alr/gfer)gen ethera p

4、euticeffect mediatedbya no velminicirclevector(mc-halr).theresultsinliver?broticratsthatr eceivedmc-halrthroughhydrod yn amics-basedtra nsfection(hbt)for8weeksindi catedthattheminicirclednavectorproducedamoreeffectivege netherapyeffectth an traditio nalplasmids(pcdna3.1halr).eve nwhe nwereducedthetr

5、eatmentdos eofmc-halrto30%(w/w)a ndthetreatme ntfreque ncyfromweeklytobiweekly,th einvitroa ndinvivoresultsstilldemonstratedthathigheralrge neexpressio nsigni? cantlyblockedincreasesi ntransforminggrowthfactorbl(tgfbl),plateletderived growthfactor-bb(pdgf-bb)/anda-smoothmuscleaorta(a-sma)levels;effe

6、ctively suppressedtheproductio no fcollage nsespeciallycollage nl;andeffectivelyalleviat edliverinjurya nd?brosisi nratsherebyimprovi ngthesurvivalrateofliver?broticrat s.ltispreliminarilyc oncl udedthattherelativeoverexpressionofmchalrinhibitsth eactivati ono fhepaticstellatecells(hscs),therebyalle

7、viati ngliver?brosisinrats keywords:minicirclednavector,high-levelexpression,augm en terofliverrege nera tion(alr),ge netherapy川 ver?brosis,hydrod yn amics-basedtra nsfection(hbt)introductionliverfibrosisresultsfromchronicdamagetotheliverinconjunctionwiththeex cessiveaccumulati ono fextracellularmat

8、rix(ecm)proteins,whichisacharacteristic ofchronicliverdiseasescausedbyviralautoimmunedrug-relatedholestaticorm etabolicdiseases.advancedliver?brosisresultsincirrhosisjiverca ncer;andliverfail ure,whichisamajorcauseofmortalityworldwide.lgen etherapyisarelativel yn ewparadigminmedicine,withe normousth

9、erapeuticp ote ntial.a ndliver-targetedge nedeliveryhasattractedagreatdealofatte ntion.21 na dditio ntowellk no wngrowthfactorszsuchashepatocytegrowthfactor(hgf)a ndep idermalgrowthfactor(egf),aug-menterofliverrege neration(alr),alsok nown asgr owthfactorervl-like(gfer),isanothercyto-kin eofvitalimp

10、orta ncetoliver3,4alrisamemberofthe newlydiscoveredalr/erv lproteinfamilywithfad-linkedsulfhydryloxidaseactivitythatcatalyzesdisul?debo n dformatio n.5,6thecxxcmotifofalrisesse ntialforcellsurvival7a ndthebioge nesi sofcytosolicfe/sproteins.8ln-deed?alrhasbee ndem on stratedtofunctio nasasur vivalfa

11、ctorforhepatocytes,a ndthedepleti ono falrproteinbya ntise nseolig onucle otidesleadstohepatocytecelldeath.7alrpromotesthegrowthofhepatocytesinth eregeneratingorinjuredliver,3,7playsapivotalroleasanimmunoregulatora ndasa n enzymeactivator,9?10andprotectshepatocytesagainstapoptosis.4furthermore,v ari

12、ousreportsi ndicatethatalrappearstobea no velimporta ntcytokinewithpote nti alcli nicalsig ni?cancei nthege netherapyofliver?brosis.!x12*corresponde nce:prof.s on gyoujnstituteoflifescie ncea ndbio pharmaceutics,s henyangpharmaceuticaluniversitno.loswenhuaroadjlooieshenyanghi n a.e-mail:yous on g206

13、;orprof.xiangpingk on g,keylaboratoryofliverdi seasezcentreofl nfectiousdiseases,458thhospitalofpla,gua ngzhou,chinaemaii:xia ngping_k on g880jhumangenetherapwlume27numberlla2016bymarya rmliebertjnc.d0l:10.1089/hum.2016.006 minicircle-mediatedalrgenetherapy881nevertheless,o neofthemajorbarrierstoalr

14、ge netherapyisthesafetya ndef?ci enc yofthevectorsusedforge netra nsferi neukaryoticcells.comparedwithviralvectors, non viralvectorsaresafeandsimpletopreparea ndmodify.l3plasmiddnaisfreque n tlyusedfor non viralge netherapy,butthepresen ceofcpgmotifsa ndtheiras-sociat edsequencesi nbacterialdnacause

15、sanimm uno toxicresponseafterthedeliveryoft heseplasmidvectorstomammalia nhosts,whichisapote ntialriskforsafecli nicalapp licatio nsan dreducestra nsgeneexpressio n.l4moreover;theresultsofourprevious experime ntdemo nstratedlowlevela ndshorttermexpressio ninresp on setoplas mid mediatedalrge nethera

16、pyi nrats,withlargei ndividualdifferences(unpublish edj.there-forethedevelopmentofa nef?cie ntan dsafedeliverysystemisessentialf oralrge netherapyminicircles(ivics),novelplasmiclbasedvectorsthatarecompo sedsolelyofeukaryoticsequencesd ono tcarryeitherantibioticresista ncege nesorb acterialorigi nsof

17、replicati on ;thus,mcsi nducemarkedlyreducedimmu nean din?a mmatoryresp on sesandeffectivelyprotecttransgeneexpressio nfrombeingsile nee dbecauseoftheprese nceofbacterialbackb on ese q uences15theuseofmcsisa nin terestingap-proachtoi ncreasethesafetya ndef?ci en cyofplasmid basedvectorsfor clinicalg

18、e netherapy.16therefore,i nthepresentstudy,a nalrexpressi on cassettei naminicirclednavector waspre-paredforthe?rsttime,theexpressi on levelofthealrge nemediatedbyami n icirclednavectorwascomparedwiththatofc onven tionalplasmidsbothinvitroa ndi n vivoa ndthe8weekge netherapyeffectin?broticratswascom

19、pared.materialsandmethodspare ntalplasmidc on structio nthehuma nalrgeneexpressi onf ragmentcontainingacmvpromoter,huma nalrc odin gseque nce(ge nban kaccessi onnu mber:nm_005262)?a ndbghpolyade nylati onsignalsequencewaspcr-ampli?edwithaforwardprimer(5 c -gactgaagatctatgtacgggccagatata-3 c )?revers

20、eprimer(5 c -ataccgctcgagggttctttccgcctcagga-3 c )zandprimestarmaxdnapoly-m erase(takara),usingthepreviouslyc on structedpcdna3.1-halr12asatemplate.th eampli?edfragme ntswereexcisedwithbgllla ndxhol(neb)a ndthe nligatedbetwee nattbandattpsites(fig.la,parentalplasmid).plasmidpreparatio napreviouslyre

21、portedprotocoll7forminicirclednavectorproductio nwasusedwit hsomemod-i?cations.brie?y,escherichiacolistrainzy-cy10p3s2t(akindgiftfromdr.zhiyingch en ,sta nforduniversity)wastra nsformedwiththepare ntalplasmids. on etra nsform an twaspickeda ndgrow no ver night(od6004.5-5.0)i n5mltbbroth(ln vitroge n

22、)con tainin gka namyci n( 50lg/ml)at37°cwithshaki ngat250rpn%followedbyampli?catio n with0.1-0.3%oftheinoculumintbbrothc on tainingka namycin(50lg/ml)for：15hr then,aftertheadditio no fal.2-foldvolumeoflbbroth(l nvitroge n)con taining0.03% l-arabi no se(sigma-aldrich)a nd3%lnnaoh,thei ncubati on wasc ontinu edfora nad ditional6hrat32°cwithshakingat250rpm.e.colistrai ndh5a(l nvitrogen)wastransformedwithpcdna3.1halr.onetransfo rmantcolonywaspickedandgrow no vernight(od6004.5-5.0)i n5mllbbroth(l nvitr oge