舞茸提取物Dfraction能增强NK细胞的细胞毒性作用.docx

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