舞茸提取物Dfraction能增强NK细胞的细胞毒性作用.docx
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舞茸提取物Dfraction能增强NK细胞的细胞毒性作用.docx
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舞茸提取物Dfraction能增强NK细胞的细胞毒性作用
声明:
本文摘自OncologyReports,由希腊知名的科技出版社SpandidosPublications出版发行,创刊于1994年,月刊。
该杂志内容覆盖肿瘤领域广泛的基础和应用研究,注重于发表肿瘤发生、转移和流行病学方面的原创性论文和综述。
为方便广大读者阅读,我们对论文摘要进行了中文翻译,译文仅供参考,如有不足之处,敬请指正。
-----本文摘自舞茸D-fraction官方网站,转载请注明
【参考译文】
EnhancementofcytotocicityofNKcellsbyD-Fraction,
apolysaccharidefromGrifolafrondosa
舞茸提取物D-fraction能增强NK细胞的细胞毒性作用
摘 要
在先天免疫中,在不受MHC抗原限制的情况下被激活的自然杀伤(NK)细胞能攻击和消灭病原体如细菌、病毒。
有报道称被IL-12激活的NK细胞在穿孔蛋白介导的细胞凋亡中能识别和杀死肿瘤细胞。
我们曾报道,一种从舞茸中提取的多糖D-fraction能激活巨噬细胞、树突细胞以及T细胞,并能抑制肿瘤生长。
尽管如此,舞茸D-fraction在先天免疫反应中对NK细胞功能的影响并不清楚。
在此项试验中,MM-46乳腺癌荷瘤小鼠连续三天腹腔内注射舞茸D-fraction,从而研究其对NK细胞活性和细胞毒性的作用。
结果显示D-fraction能显著增强NK细胞对于NK敏感性靶细胞YAC-1细胞的细胞毒性以及其CD223的表达量。
D-fraction也能增加巨噬细胞的CD86表达量。
另外,与对照组相比,脾细胞培养上清液以及血清中的IL-12量也增加,相应地NK细胞中IL-12受体β1的表达量也增加。
这些结果表明D-fraction通过激活巨噬细胞分泌IL-12来增强NK细胞的细胞毒性。
关键词:
NK细胞,细胞毒性,白介素-12受体
论文原文
ONCOLOGYREPORTS13:
497-502,2005
EnhancementofcytotoxicityofNKcellsbyD-Fraction,
apolysaccharidefromGrifolafrondosa
NORIKOKODAMA1,AKIHIROASAKAWA2,AKIOINUI2,YUKIMASUDAandHIROAKINANBA1
1DepartmentofMicrobialChemistry,KobePharmaceuticalUniversity,4-19-1,Motoyama-kitamachi,Higashinada-ku,Kobe658-8558;2DivisionofDiabetes,DigestiveandKidneyDiseases,DepartmentofClinicalMolecularMedicine,KobeUniversityGraduateSchoolofMedicine,7-5-1Kusunoki-cho,Chuo-ku,Kobe650-0017,Japan
ReceivedAugust10,2004;AcceptedOctober4,2004
Abstract. Ininnateimmunity,activatednaturalkiller(NK)cellsattackanddamagepathogenssuchasbacteriaandviruswithoutrestrictionbytheMHCantigen.NKcellsactivatedbyIL-12havebeenreportedtorecognizeandkilltumorcellsinperforin-mediatedapoptosis.WehavereportedthatD-Fraction,apolysaccharideextractedfromthemaitakemushroom(Grifolafrondosa),activatesmacrophages,dendriticcells,andTcellsandinhibitsthegrowthoftumorcells.However,theeffectsofD-FractiononNKcellfunctionintheinnateimmuneresponsearenotwellknown.Inthepresentstudy,weadministeredD-FractiontoMM-46mammarytumor-bearingC3H/HeJmiceintraperitoneallyfor3consecutivedaysandinvestigateditseffectsontheactivationandcytotoxicityofNKcells.D-FractionsignificantlyenhancedthecytotoxicityagainstNK-sensitiveYAC-1cellsandtheexpressionofCD223onNKcells.D-FractionalsoincreasedtheexpressionofCD86onmacrophages.Inaddition,thelevelsofIL-12intheculturesupernatantofwholespleencellsandinserumincreased,comparedwiththecontrolcorrespondingtoanincreaseinexpressionofIL-12receptorBIonNKcells.TheseresultssuggestthatD-FractionenhancesthecytotoxicityofNKcellsthroughtheproductionofIL-12bymacrophagesactivatedbyD-Fraction.
Keywords:
naturalkillercell,cytotoxicity,interleukin-12receptor
Introduction
Naturalkiller(NK)cellsareresponsibleforinnateimmunityandhavetheabilitytorecognizeandkilltumorcellswithoutcompletelyexpressinghistocompatibilitycomplex(MHC)classIandIIantigens,bywhichNKcellsaredistinguishedfromBandTcells
(1).DespitesomesimilaritiestoTcells,NKcellsarenotdependentonthethymusfordevelopment,andlacktheTcellantigenreceptorandexpressionoftheCD3complexonthesurface(1,2).RestingorunstimulatedNKcellsconstitutivelyexpressanumberofcytokinereceptorsandmaybeactivatedforincreasedcytolyticactivityorcytokineproductionfollowingstimulationbynumerouscytokines(e.g.IL-2,IL-12,IL-15,IL-18,andIL-21)orIFN-α/β.NKcellsareunabletoproduceanyofthesecytokines
andaredependentonothercelltypestoprovidethesefactors
(1).TheactivationofNKcellsbythesesignalfactorsorcognateinteractionswithtumorcellsalsoinducesNKcellstotranscribeandsecretecertainchemokinesandcytokinesincludingIFN-γ,GM-CSF,G-CSF,M-CSF,TNF-α,andothers(3,4).
IL-12,aheterodimericcytokineproducedmostlybyantigen-presentingcells(APCs)suchasstimulatedmacrophages,monocytes,dendriticcells,neutrophils,andsomeBcellsinducescytokineproduction,primarilyofIFN-γbyNKandTcells,enhancesthecytotoxicactivityofNKcells,andpromotesdifferentiationofnaiveThcellsintoThlcells(5-7).Thus,IL-12representsafunctionalbridgebetweentheearlynon-specificinnateresistanceandsubsequentantigen-specificadaptiveimmunity.
NKcellshavealsoshowntobeactivatedbyIL-12tokillvarioustumorcellsinaperforin-dependentmanner(8).Thepore-formingproteinperforinisakeyeffectorproteinforcytolysismediatedbyNKcellsandcytotoxicTcellsandispreferentiallyexpressedinthesecells.Perforin-mediatedapoptosisistheprinciplepathwayusedbyNKcellstokilltumorsandvirus-infectedcells.AlthoughNKcellsconstitutivelyexpressperforinatalowlevel,IL-12andIL-2havebeenshowntoinduceincreasedaccumulationofperforinmRNAinNKcells(9).ThebiologicalfunctionsofIL-12aremediatedthroughspecificreceptorsonNKandTcells.TwoIL-12receptorsubunits,IL-12receptorβl(IL-12Rβ1)andIL-12receptorβ2(IL-12Rβ2),havebeenidentifiedinmouseandhuman(10-12).IL-12specificallyactivatesSTAT4inNKandTcellsbyinducingtyrosinephosphorylationthroughactivationoftheIL-12receptor-associatedJAKfamilykinases,Jak2andTyk2(13).
Mushroomshavebeenvaluedasflavorfulfoodsandmedicinalsubstancesandarewidelysoldasnutritionalsupplements.Polysaccharidesfrommushroomshavebeenreportedtoenhancetheimmunesystem.LentinanfromLetinusedodes,schizophyllan(SPG)fromSchzophyllumcommune,SSGfromSclerotiniasclerotiorum,andPSKfromCoriolusversicolorpotentiallyexertantitumoreffects(14).Wepreviouslyreportedthatthe(l→3)-branched(l→6)-β-glucan,termedtheD-Fractionandextractedfromthefruitingbodyofthemaitakemushroom(Grifolafrondosa),aswellasthesepolysaccharides,isabiologicalresponsemodifier(BRM),enhancedanimmuneresponsethroughtheactivationofAPCsandTcells(15-17).TheadministrationofD-FractiontoMM-46tumor-bearingC3H/HeJmiceorcolon26tumor-bearingBALB/cAmiceinducedaTh-1-dominantresponsethroughIL-12productionbyactivatedAPCs(18,19).
IL-12hasbeenshowntopossesspotentantitumoractivityinawidevarietyofmurinetumormodels,andtheactivityhasbeendemonstratedagainsttumorsofvarioushistologies,includingtumorsarisingfromthecolon(CT26),kidney(Renca),andlung(3LL);carcinogen-inducedsarcomalines;andmelanoma(B16F10)(20-24).Inthepresentstudy,weinvestigatedwhetherD-FractioncanenhancetheactivationandcytotoxicityofNKcellsintheinnateimmuneresponse
tothetransplantationofMM-46mammarytumorcellstomice.TouseD-FractionforcancertreatmentwithIL-12,weinvestigatedtheexpressionofIL-12RβlonNKcellsbyflowcytometryandsuggestedwhetherD-Fraction-inducedIL-12enhancesthecytotoxicityofNKcellsbystimulatingtheIL-12signalingpathwayinNKcells.
Materialsandmethods
Animals.LPSnon-responsivemaleC3H/HeJmice(4weeksold)(CLEAJapan,Inc.,Tokyo,Japan)wereraisedfortwoweeksbeforebeingusedforexperiments.Foodandwaterwereprovidedfreelytothesemiceuntiltheexperiments.
Tumorcells.MM-46(C3H/Hemouse-derivedmammarytumor)cellswerekindlydonatedbyDrKankiKomiyamaandmaintainedintheperitonealcavityofmaleC3H/HeJmice.ThemurinelymphomacelllineYAC-1(ATCCnumber:
TIB-160)waspurchasedfromDainipponPharmaceuticalCo.,Ltd.(Osaka,Japan)andmaintainedinRPMI-1640mediumsupplementedwith10%heat-inactivatedbovineserum(FBS).
PreparationofD-Fraction.D-Fractionwaspreparedfromthedriedpowderofthefruitingbodyofmaitakemushrooms(Grifolafrondosa)(YukiguniMaitakeCo.,Ltd.,Niigata,Japan),accordingtothemethoddescribedinourpreviouspaper(15).ThelevelofLPScontainedinD-FractionwasdeterminedusingEndospecyES-20SSet(SeikagakuCorp.,Tokyo,Japan),andwas<0.0000006%.
AdministrationofD-Fraction.MM-46tumorcells(l×l06)wereimplantedintherightaxillaryregionof6-week-oldmaleC3H/HeJmice.After24h,4mg/kgperdayofD-Fraction,4mg/kgperdayofdextran(molecularweight:
580000,Sigma-AldrichCorp.,St.Louis,MO,USA),orsalinewasadministeredtoMM-46tumor-bearingmiceintraperitoneally(i.p.)onceadayfor3or7consecutivedays.Onday4,tumorvolumeswerecalculatedaccordingtotheformula:
(tumorvolume)cm3=(longestdiameter×shortestdiameter2/2).
Preparationofwholespleen,splenicNKandwhitebloodcells.After3daysofD-Fraction,dextranorsalineadministration,aspleencellorbloodcellsuspensionwasprepared.Erythrocytes,deadcellsanddebriswereremovedfromeachcellsuspensionusingLympholyte-M(CosmoBioCo.,Ltd.,Tokyo,Japan),andwholespleenorwhitebloodcellsweresuspendedinRPMI-1640mediumcontaining0.5%FBS.SplenicNKcellswerepreparedfromwholespleencellsusingtheMACSnegativeselectionsystem(MiltenyiBiotecGmbH,BergischGladbach,Germany).ThepurityofsplenicNKcells(CD49b/Pan-NK+CD3-cells)wasconfirmedbyflowcytometricanalyses,andwas>89%.
Cytotoxicityassay.Cytotoxicityassayswereinvestigatedbymeasuringandevaluatinglactatedehydrogenase(LDH)releasefromlysedYAC-1cell(25).SplenicNK,wholespleen,orwhitebloodcellswereincubatedwithYAC-1cellsfor4h(E:
T=25:
1forsplenicNKcells,50:
1forwholespleencells,and50:
1forwhitebloodcells).ThelevelofLDHrelea
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- 提取物 Dfraction 增强 NK 细胞 毒性 作用