氧化苦参碱对小鼠淋巴结T细胞增殖的双向作用

氧化苦参碱对小鼠淋巴结T细胞增殖的双向作用 氧化苦参碱对小鼠淋巴结T细胞增殖的双 向作用 第16卷第4期 2006年2月 中国现代医学杂志 ChinaJournalofModernMedicine Vol_16No.4 Feb.20()6 文章编号:1005—8982(2006)04-0492-05 ? 论着? Two-wayseffectsofOxymatrineonTlymphocyteproliferation inmouselymphnodestimulatedbyConA WUBin,XIEHong-fu,LILuo-si,ZHANGJiang—lin,LIJian-guo f.DepartmentofDermatology,2.InstituteofBiomedicalEngineering,XiangyaHospital, CentralSouthUniversity,Changsha,Hunan4JOOO&P.R.China) Abstract:【0bjectlve】 ToinvestigatetheeffectsofOxymatrine(0M,r)onmouselymphocyteproliferationstimu- latedbyConA,explorethemechanismoftheeffeetsofOMTontheimmunesystemandprovidetheoreticalandex— pefmentalevidencefortheclinicalapplicationofOMTintreatingimmune—relateddiseases. 【Methods】CFDA—SE stainingandflowcytometrywereusedtodetectthefluorescenceintensityoflymphoeytesafterstimulatedbypoly? elonalstimulatorsConAandOMT.RelatedsoftwareswereusedtoanalyzetheeffectsofOMTonmouselymphocyte proliferation.【Results】 OMThasthefunctiontorestraintheproliferationoflymphocyteofmousedependedonits concentrationwith500,125and31g/mLinsubstrate,butl6,8,4,2mLconcentration,itimprov estheprolif- erationofTlymphocytesofmouse'Slymphnode,thedependenceonitsconcentrationisnotsignificant.【Conclu- sions】1.BothCFDA— SEdyeingandflowcytometerwerereliabletoolstoanalyzelymphocyteproliferation.2.OMT hasthetwo— wayseffectsonTlymphocyteproliferationinmouselymphnodestimulatedbyConA. Keywords:CFDA-SE;Oxymatrine(OM,r);lymphocyte;proliferation CLCNumber:R34Documentcode:A 氧化苦参碱对小鼠淋巴结T细胞 增殖的双向作用 伍斌,谢红付,李罗丝,张江林,李建国2 (中南大学湘雅医院1.皮肤性病科;2.生物工程研究院,湖南长沙410008) 摘要:目的检测氧化苦参碱(Ox-ymatrine,OMT)对刀豆蛋白A(ConA)刺激的小鼠淋 巴结T细胞增 殖的影响,探讨OMT对免疫系统的作用机制,为临床用OMT治疗免疫相关性疾病 提供理论和实验依据.方 法利用CFDA—SE染色,流式细胞术检测淋巴细胞在多克隆刺激剂ConA和OMT 的共同作同下荧光强度 的变化,并应用CELLQuest软件 分析 定性数据统计分析pdf销售业绩分析模板建筑结构震害分析销售进度分析表京东商城竞争战略分析 OMT对小鼠淋巴结T细胞增殖的影响程度. 结果500,125和 31g/mLOMT对小鼠淋巴结T细胞增殖呈剂量依赖性抑制,而16…842g/mLOMT 对小鼠淋巴结T细 胞增殖起促进作用,但其剂量依赖关系不明显.结论CFDA-SE染色和流式细胞术 是分析淋巴细胞增殖的有 力工具;OMT对小鼠淋巴结T细胞的增殖呈双向作用. 关键词:CFDA-SE;氧化苦参碱;淋巴细胞;增殖 中图分类号:R34文献标识码:A Oxymatrine(OMT)isthemajorvalidalkaloidthat extractedfromSophorofiavescensAit,akindoftra— ditionalChinesemedicine.Researchesdemonstrate thatithasasignificantfunctionofadjustingthedif- Receiveddate:Jan.20,2006 【Correspondenceto】XIEHong-fu,E-mail:XieHongfu@Tom.con ferentiationandproliferationofTandBcellsin spleenofmouseandlymphokinegeneration,itcanal— SOobviouslyrestrainthetypeI—IVallergies【lJ_Howev— eLthereisfewrepotsinChinaabouttheeffectof - 492- 第4期伍斌,等:氧化苦参碱对小鼠淋巴结T细胞增殖的双向作用 OMTonthegenerationofmiceTlymphocytes. Carboxyfluoresceindiacetate,succinimidylester fCFDA—SE)isafluorochromeofcellularstructurepro— tein.Itcanconstantlyremaininalivingcellformore than2weeksandevenlydistributedintofilialgenera— tioncells,soflowcytometryfordetectingthefluores— cenceintensitycanbeusedtoanalyzethenumerical distributionofceUintheeachfilialgenerationcells[z3】. UsingCFDA-SEstainingandflowcytometrytostudy theeffectofOMTonthecellproliferationoflympho— cytestimulatedbyConcanavalinA(ConA).Itex— plainsthemechanismoftheeffectsofOMTonthe immunesystemandprovidestheoreticalandexperi— mentalevidencefortheclinicalapplicationofOMTin thetreatmentofdiseasesrelatedtoimmunity. 1Methods J.1Preparationoflymphocytesuspension Balb/cMousewaskilledwiththebreakdownofits spinalcordandbothsuperficialandmesentericlymph nodesfromaxillae,infra—claviclesandfoldinguen wereasepsissegregatedandthetegumentswerere— moved.Thenfilteredwithnylonmeshsieveat200一 meshscreensothecellswerecollected.Thecells werewashedwithPBSandcentrifugalized(300g,5 min1for2timesandkeptinPBSinsuspension. J.2CFDA-SEdyeingoflymphocyte CEDA—SEweredissolvedwithdimethvlsulfl0xide (DMSO)into10mmol/Ldepositfluidandkeptina一 20qCcondition.Beforeusing.appropriatesumofthe fluidweretakenandfurtherdilutedwithPBSinto 21xmoL/Lasworkingfluidandequilibratedtothe roomtemperatureforuse.Thedensityofthecellsin lymphocytesuspensionwerealsoadjustedto2xl0加 cells/LandequalCFDA—SEfluidfwithfinalconcen— trationof11zmol/L)wereaddedinandmixedwell, thenoscillatedanddyedsoftlyinroomtemperature f0r10minutes.Thefluidofcompoundwereagain washedwithPBSandcentrifugalized(300g,5min)2 times.andfloatedinRPMI—l640completecultureso— lutionthatcontainedl00mL/LFBS.Thecelldensity wasalsoadiustedto2xlOcells/L. J.3EffectofOMTonlymphocyteproliferationof mouse TheabovelymphocytesthatlabeledwithCFDA— SEwereincubatedin24-wellcellcultureplatesat lmL/wel1.Wesetcontrolgroup,ConAgroupandCon A+OMT500咖Lgroup,ConA+OMT125Ixg/mL group,ConA+OMT31p4/mLgroup,ConA+OMT16 p,g/mLgroup.ConA+OMT8p,g/mLgroup,ConA+ OMT4咖L,ConA+OMT2Ixg/mLgroup.Allcul— turesweremadeintriplicated.Thefinalconcentration ofConAintheabovegroupswas10咖Lequally. Underaconditionof37?.5%CO2,allcultureswere analyzedforCFSEfluorescenceintensity(FL1)atthe timeofculturedfor48hand72hrespectively. AIldatawereacquiredviaFACSCaliburCytometer andthenanalyzeditinCELLQuestsoftware.Thelyre— phocyteregionsR1inthebi——dimensionalscatter..plot offorwardscatter(FSC)andsidescatter(SSC)were singledoutfortheFL1intensitytestofeverygroup. Eachtubesamplewilldetect10000cellsandthe datawereanalyzedbyCELLQuest. 2Results 2.JEffectsofOMTontheproliferationofTlym- phocytesofmouseintherangeof31-500ttg/mL Aftercuhured48h.CIEfluorescencecouldbe examinedbycytometer,whilecontrolgroupdidnot appearsub-peaks,justshownaboriginalpeak(Figure 1A).Itwasalsotosaythatlymphocytesdidnotpro— liferate.ThreepeakswereobviousinConAgroup, suchasfigure1B,thethreepeaksfromrighttoleftare aboriginalpeak,1stfilialgenerationpeakand2ndfil— ialgenerationpeak.ThismeansthatLymphocytesap— pearedobviouscleavageafter48hstimulatedbyCon A.comparewithcontrolgroup,fluorescencedensityof CFSEofeachgenerationhalvedclearly.Inallgroups ofConA+OMTtreated,highdoes(OMT500Ixg/mL) groups(Figure1B),Aboriginalpeakismuchhigher thanthatofConAgroup,butthe1stand2ndfilial generationpeakismuchlowerthanthatofConA group,whilecomparetocontrolgroup,fluorescence densi~ofCFSEofaboriginalpeakdecreaseslightly, namely,thereisnoobviousproliferationoflympho— cyte,thisimpliesthatOMTconstrainthelymphocyte proliferationsignificantly.Theresultsofmiddlingdose (OMT125p4/mL)group,asFigure1C,alsoshowed lessobvious1stand2ndfilialgenerationpeak,but ? 493? 中国现代医学杂志第l6卷 comparedwithConAgroup,aboriginalpeakismuch higherand1stand2ndfilialgenerationpeakismuch lower.ThisshowedthatmiddlingdoseofOMTcould alsoobviouslyrestrainmouselymphocyteproliferation. Inlowdose(OMT31~g/mL)group(Figure1D),ap— pearedtwoveryobvioussub-peaks,comparedtoCon Agroup,ithasnosignificantdifferences.Butcom: paredtocontrolgroup,thefluorescenceintensitiesof resultsofallgroupshadsignificantdifferences.It showedthatOMTofthisdosecouldntsignificantly restrainlymphocyteproliferation.After72hculture, thefluorescenceintensitychangedbetweenallgroups consistenttothoseofculture48h.Controlgroup (Figure2A)justshowedsinglepeak,itmeansnolym. phocyteproliferation.Muhi—sub-peakinConA groupsshowedlymphocyteproliferationevidently, suchasFigure2B.IntheConA+OMT500~g/mL aboriginalpeakswerehigherthantheConAgroup, butallthesub——peakswerelower.ThismeansOMT stillrestrainlymphocyteproliferationafter72h.Abo- riginalpeaksandthesub——peaksoftheConA+OMT 125~g/mLgroupFigure2C)showedOMTstillre— strainlymphocyteproliferation.Thedifferenceswere unconspicuousbetweenthepeaksofConA+OMT31 Ixg/mLgroup(Figure2D)andthepeaksofConAgroup, butthedifferenceswereclearbetweenthisandcon- trolgroup.Insummary.nomatterculturedf0r48hor 72hwithOMTrespectivelyin500Ixg/mL,125Ixg/mL ConA+SB~Ilg/mL, . ^ 1ir _,_. and31Ixg/mLcouldrestrainedthepromotioneffectof ConAonmouselymphocyteproliferationinmanner ofdosedependence. 2.2EffectsofOMTontheproliferationofTlym- phocytesofmouseintherangeof2~16ttg/mL After48hoursculture,theCFSEfluorescencein— tensityofeachgenerationofceilswasgotintheflow cytometrytest.Inthecontrolgrouptherewasnofilial generationpeaksbutonlyshowedaprimarygenera. tionpeak(Figure3E).IntheConAgroup,itap— pearedthreeobviouspeaks,theywere(fromright side)primarypeak,1stfilialgenerationpeakand2nd filialgenerationpeak.Itclearlyshoweddifferencesin cellfluorescenceintensityineachgenerationascom— paredwiththoseofthecontrolgroup.IneachConA十 0MTgroups.wecouldseedistinctresultsInOMT16 ~g/mLgroup(Figure3A),therewerenosignificant differencesoffluorescenceintensityinprimarygener— ationpeakandthe2ndfilialgenerationcompared withthoseoftheConAgroup,butthe1stfilialgen— erationpeakobviouslyhigherthanthatoftheConA groupwhiletheprimarygenerationlowerthanthatof thecontrolgroup.InOMT8Ixg/mLandOMT4~g/mL groups(Figure3B,3c),itappearedveryclearly1st and2ndsub—peaks.ComparedwithConAgroup,the primarygenerationpeakswerelowerbutthe1stand 2ndsub——peaksweremuchhigherthanthoseinConA groupwhichshowedstatisticalsignificance.InOMT2 1Fluorescenceintensityoflymphocytesineachgroupaftercultureof48h Figure2Fluorescenceintensityoflymphocytesineachgroupaftercultureof72h ? 494? 第4期伍斌,等:氧化苦参碱对小鼠淋巴结T细胞增殖的双向作用 ~g/mLgroup(Figure3D),theprimarygenerationpeak showedlessdifferencewiththatofConAgroup,but the1stand2ndsub——peakswereslightlyhigherthan thoseofConAgroupsandshowedsignificantdiffer- encescomparedwiththoseofthecontrolgroup.After 72hculture,thefluorescenceintensityofallgroups areconsistentwiththoseofculturedfor48h.In"con trolgroup(Figure4E),theyalsoshowedsinglepeak, andnocellproliferation (Figure4A),itappeared wasfound.InConAgroup severalfilialgenerationpeaks whichshowedcellproliferationclearly.Amongthe ConA+OMTgroups,italsoshowedsomedifferences. InOMT16mL,8~g/mLand4~g/mLgroups (Figure4A,4B,4C),theprimarypeakobviouslylower thanthatofConAgroup,whilethefilialgeneration peaksweremuchhigherthanthoseoftheConA groups.InOMT2~g/mLgroup(Figure4D).there werenodifferencesinallsub—peakscomparedwith thoseoftheConAgroup,butshowedcleardifference withthoseofthecontrolgroup.Aftercuhuredfor72 hours,OMTstillpromotedtheproliferationofmouse lymphocytesignificantlyandthepromotionshowed lessdependenceonthedosage.Ingeneral,OMTcan promotetheproliferationofmouselymphocyteand showedlessdependenceonthedosageinthevarious 0MTgroups. Figure3Fluorescenceintensityoflymphocytesineach groupafterculturedfor48h R{蚋 \C黜0l Figure4Fluorescenceintensityoflymphocytesineach groupafterculturedfor72h 3Discussion Oxymatrine(OMT)isanalkaloidextractedfrom therootsandseedsofSophoroflavescensAir.Plenti— fu1documentsofpharmaceutical,andclinicalresearch showedthat0MThadthefunctionsofanti-inflamma— tory,anti—virus,anti-hepatic-fibrosis,liver—protec— tion.enzyme—lower;anti—cancerleucocyte—promotion, anti—arrhythmiaandotherpharmaceuticalproperties, whilehadlessside—effectst4~.OMTiScommonlyused intreatingdermatitis,eczema[71,arrhythmiaandmalig— nanttumor[ , aswellaswidelyusedtotreattypeB hepatitistg~.Theyallhadgoodtherapeuticeffect,but themechanismofthefunctionsremainedunclear.Our studyfoundthatOMTcanconstraintheproliferation ofmouselymphocytesanddependingonitSconcen trationfrom31500~g/mLinthesubstrate,while ? 495? 嚣嚣2糟 量芒《 tx 搴窑鲁. ,^v 是异6 S ?2#? - 中围现代医学杂志第16卷 range2~161~g/mL,OMTclearlyshoweditseffectson thepromotionofthemouselymphocyteproliferation withoutdistinctdependenceonthedosage.Thehis— togramsrefertoFigure3andFigure4.Allthisindi— catestheregulationtoautoimmunityandoffersclue forthemechanismofOMTtreatingtype——Bhepatitis inclinic.Therearesomestudiesshowedthatithas somecomparabilitytothisstudythatOMThasthe functionofraisingthedensityoftheCD4Tcellsin peripheralbloodofthepatientswithtypeBhepatitis anddecreasingthedensityofCD8+Tcellt砌.restrain— ingtheformationofTGF—Blandpreventingliver-fi— brosis",whenatalowerdosage,OMTcanstrengthen theproliferationoflymphocytesandthegenerationof IL一2,thisexistsacomparabilitytoourstudy.Besides, OMThastheobviousanti-canceractivity,itshows significantconstrainingabilityonsarcoma$180and ascites-transplanted—sarcomaofmouse180At21;italso couldrestrainthedevelopmentoftumorcellofliveB, stimulatethesecretionofIL一2.raisetheNKcells activity.activatetheimmunecellI4.OMT1.25mg/kg showedobviousefficacyonmouseEhrlichscarcino. ma($18o),itschemotherapeuticalindexis7.8times ofthatofmitomycin.0MT375mg/kgcanprolongthe lifespanofmousewiththeascites—transplanted—can— cerfor129.9%.OMTalsoobviouslyrestraineduterine cervixcancerU14ts~,italsoshowedsignificantrestrain onproliferationofvascularendothelialcell(VEC)that inducedbythecellsofbronchogeniccarcinomaand gastriccancer.AlltheseshowedthatOMTprobably becomeaclinicaltreatmentmedicineforpreventionof themetastasisoftumors,andwillbeoneofthevery promisingnon-killingtreatmentsforthecancer[~lZ. inaddition,OMTcanalsoconstraintheactivationof APCcells.inducetheapoptosisofkeratinocytes【4】 andsoon,allthishassomecomparabilitytothis study.InvirtueofOMTconstraintheproliferationof lymphocytes.andthatallkindsofimmune—related diseasessuchassystemiclupuserythematosus,aller— giccontactdermatitis,atopydermatitis,eczemas,pso— riasisandsoon.allthesediseasesarerelatedtothe activationandproliferationoflymphocytesdirectlyor indirecdy'Therefore.maybethisisoneofthereasons oftheeffectofOMTintreatingthesediseases.Be— sides.thisresearchshowsthetwo—wayeffectsonthe proliferationoflymphocytestimulatedbyConAthat canbedetectedthroughthechangesofCFSEfluores- cenceintensities.ThroughCELLQuestsoftwarewea1. sogetlotsofrelatedindexofproliferationandthuswe havefulfilledvisualandnumerousstudyoftheeffect ofOMTontheproliferationoflymphocytes.Compared ~kiththetraditionalproliferationtestingapproaches,it showsgreatadvantages.Westudiedtheeffectsof OMTontheproliferationoflymphocyteswithsemi- quantitativemethod.Undertheconcentrationsof500, 125,31mL,OMTclearlyshoweditsobviousre— straining——effectsontheproliferationofthemouse lymphocytewithadistinctdependenceontheconcen— trationinthesubstrate.ThehistogramsrefertoFigure 1andFigure2.whileundertheconcentrationsof16, 8,4,2I~g/mL,OMTclearlyshoweditseffectsonthe promotionofthemouselymphocyteproliferationwith— outdistinctdependenceonthedosage.Thehistograms refertoFigure3andFigure4.Thisstudycanpartial— lyexplainthemechanismofitsimmunemodulation andrestrain—effectsoninflammationofOMT.Italso providessomeexperimentalbasisfortheclinicaluti lizationofOMTinthetreatmentofallergicdermatosis andimmune—reIateddiseases[J+4习. 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