细胞因子及其应用

细胞因子及其应用MjmacrophageIL-1Cytockinesarelow-molecular-weightregulatoryproteins

orglycoproteinssecretedbywhitebloodcellsandvariouscells(vascularendothelialcell,epidermiccellandfibroblast)inbodyinresponsetoanumberofstimuli.

ConceptofcytokineCytokinegeneInducingstimulusCytokine-producingcellCytokineGeneactivationReceptorsignalBiologicaleffectsOverviewoftheinductionandfunctionofCytokine1.interleukin,IL（白细胞介素）2.colonystimulatingfactor,CSF（集落刺激因子）3.interferon,IFN（干扰素）4.tumornecrosisfactor,TNF（肿瘤坏死因子）5.Chemokine,CK

（趋化因子）6.growthfactor,GF（生长因子）一、细胞因子的分类和功能IL-13IL-2IL-41.Interleukin,ILInterleukinsaresecretedbysomeleukocytesandactuponotherleukocytes,IL-1-IL-38havebeenidentified.2.Colonystimulatingfactor,CSFThecytokinesthatstimulatehemopoieticcellstoformcellcolony,ParticipatehemopoieticfunctionarecalledCSF,forexampleGM-CSF,G-CSF,M-CSF,Epo,Tpo.

G-CSFGM-CSFThefunctionsofCSFThecytokinesthatresistviralinfection,andinterfereviralreplication,includingIFN-α,IFN-β(typeI);IFN-γ(typeII)3.Interferon,IFNviruscellinfectionIFN-α,IFN-βIFN-αIFN-βTheanti-virusmechanismofIFN-γIFN-inducedproteinIFN-γviralreplicationInhibitionofviralreplicationnaiveCD4+TcellsAPCMicrobesProliferationanddifferentiationIFN-γMacrophageactivation(enhancedmicrobialkilling)IFNγ(typeII)EffectorTcellTheanti-MtbmechanismofIFN-γ

Mycobacteriumtuberculosis

(Mtb)

4.Tumornecrosisfactor,TNFThecytokinesthatinducedirectlyapoptosisoftumorcells,includingTNFα,TNFβ

(Lymphotoxin,T),FASL,CD70L,CD30L,CD40L,OX40L,TRAIL(TNFrelatedapoptosis-inducingligand)(1)CytokinesoftheTNFFamilyCanBeSolubleorMembraneBoundTNF-αFASLTNF-αTNF-TNFR2complex.Green:TNF;Blue:TNFR2(2)TNFReceptorsTargetcellapoptosisTargetcellactivationinflammationAcanceroustumorinamouseinjectedwithendotoxin(left)showshemorrhagicnecrosis,unlikeatumorinanuntreatedmouse(right).EndotoxininducestheproductionofTNF,whichthenactstodestroythetumor.TNFandinflammationThecytokinesthatpromotegrowthofvariouscells,epidermalgrowthfactor，insulin-likegrowthfactor，vascularendothelialgrowthfactor,transforminggrowthfactor.5.Growthfactorvascularendothelialgrowthfactor(VEGF)Epidermalgrowthfactor

GrowthfactorsappliedtoangiogenesisVascularendothelialgrowthfactor(VEGF)VEGFpromotesTumorAngiogenesisFunctionofthelymphaticvasculatureinhealthanddiseaseRoleofdrugsintheTreatmentoftumorSorafenib是一种新颖的，口服有效的RAF激酶和VEGFR抑制剂

6.ChemokineChemokines,agroupoflow-molecular-weightcytokinwsincludinginterleuki8,thataffectchemotaxisandotheraspectsofleukocytebehavior.C:CysteineCXCRandligandsduringairwayinflammation

Chemokinemaketheshapesofcellschanged对照组实验组增殖转移血管新生Chemokinereceptorantagonistintumortherapy二、细胞因子与临床Relateddiseases

：

deficiencyofcytokinesandreceptorsoverexpressionofcytokineshighleverofsolublecytokinereceptorsTherapeuticuseofcytokinesandtheirreceptors：

supplyandadditionblockageandantagonist

1.器官移植排斥反应IL-1,IL-2,TNF,IFN,IL6（一）细胞因子与疾病的发生2.免疫缺陷病IL-2,-4,-7,-9and-15Mutationsintheγc(commonγchain)chainofthereceptorsforinterleukinsIL-2,-4,-7,-9and-15**Signaltransduction,**Lymphocytedifferentiation,proliferationandmaturationInthe1990s,late

researcherstestedagenetherapytreatmentthatwouldrestorethefunctionofacrucialgene,gammac,tocellsoftheimmunesystem.Thistreatmentappearedverysuccessful,restoringimmunefunctiontomostofthechildrenwhoreceivedit."bubbleboy"disease,3.参与炎症反应细胞因子风暴ProposedMechanismoftheCytokineStormEvokedbyInfluenzavirus

CytokineStormsMayHaveCausedManyDeathsinthe1918SpanishInfluenza4.超敏反应5.自身免疫性疾病PsoriasisOverexpressionofchemokinesinpsoriasis（二）细胞因子与疾病的诊断(1)InflammatoryCytokinesandtheRisktoDevelopType2Diabetes(2)Humanneutrophilgelatinase-associatedlipocalin(NGAL),IL-18,GST-,and-GSThavegoodpotentialforearlydiagnosisofAKI,andNAG,KIM-1,andIL-18havethehighestpotentialformortalityriskpredictionafterAKI.Keratinocyte-derivedchemokine(KC)isanearlybiomarkerofischemicacutekidneyinjuryKClevelswereelevatedinserumwithin1hofrenalischemiainthemouseandincreasedinkidneytissueandurinewithin3h.Fig.1.Delayedriseinserumcreatinineafterrenalischemia-reperfusioninjury(IRI).Fig.5.ElevatedserumKCearlyafterrenalIRI.Fig.6.IncreasedurineKCearlyafterIRI.Urineinterleukin-6isanearlybiomarkerofacutekidneyinjuryinchildrenundergoingcardiacsurgeryFigureUrineIL-6isincreasedaftercardiopulmonarybypassinpediatricpatients.MousemodelsofacutekidneyinjuryFigureUrine,serum,andrenalIL-6inpre-renalazotemiaandischemicAKI.尿液、血清和肾IL-6在肾前性氮质血症和缺血性AKIFigureRenalfunctionandurine,serum,andrenalIL-6incisplatin-inducedAKI.（三）细胞因子与疾病的治疗1.重组细胞因子表1-3

已批准上市的重组细胞因子药物药物名称适应症IFN-α毛细胞白血病、肝炎、Kaposi肉瘤IFN-β多发性硬化症IFN-γ类风湿关节炎、慢性肉芽肿、生殖器疣IL-2 癌症、免疫缺陷IL-11放化疗所致血小板减少症SCF 与G-CSF联合应用于外周血干细胞移植G-CSF自身骨髓移植、化疗导致的粒细胞减少症、再生障碍性贫血GM-CSF自身骨髓移植、化疗导致的血细胞减少症、再生障碍性贫血EPO慢性肾衰导致贫血、癌症或癌症化疗导致的贫血、失血后贫血EGF 外用药治疗烧伤、溃疡（国内批准上市） TPO 放化疗所致血小板减少症（国内批准上市）2.细胞因子抑制剂表1-4

FDA批准上市的细胞因子拮抗剂和抗细胞因子抗体名称适应症可溶性IL-1R（吸入剂）哮喘可溶性IL-1R（注射剂）急性髓样白血病、类风湿关节炎可溶性IL-4R哮喘人源化抗IL-4和IL-5单克隆抗体哮喘可溶性TNF-RⅡ-Fc融合蛋白类风湿关节炎、慢性心衰可溶性TNF-RⅠ-Fc融合蛋白休克、类风湿关节炎、多发性硬化人源化抗TNF-α单克隆抗体局限性肠炎、类风湿关节炎人源化抗HER-2单克隆抗体乳腺癌人源化抗VEGF单克隆抗体晚期肺癌、肠癌ExtracellulardomainsofhumansolubleTNFreceptorHumanIgGFcfragment(stabilizesthemolecules)Etanercept(益赛普)(a–c)Psoriasis:atweek0oftreatmentwithetanercept;(d–g)Psoriasis:atweek48withetanercept1生物学活性检测;2免疫学检测;3分子生物学检测四、细胞因子的检测技术**细胞因子的特性48-WellMicroChemotaxisChamber（一）生物活性检测法—细胞因子的生物学活性趋化小室检测趋化因子Chemotaxisassays

areexperimentaltoolsforevaluationof

chemotactic

abilityof

cells.

最常用的方法是Boyden小室法，也称微孔滤膜法。趋化因子最基本的功能是引起特定细胞的趋化运动，通常用48孔的趋化小室进行实验，该小室分为两层，上层放细胞，下层放待检测因子，两层之间有聚炭膜隔开。趋化小室加细胞因子小室下层盖膜加上层小室加细胞37ºC,30分钟湿盒取下膜染色,制片Thisscanningelectronmicrographshowstwohumanmonocytesmigratingthroughtheporesofafilterinresponsetoachemokinediffusingupfrombeneath显微镜下观察,计数:细胞数/视野结果2.Transwellassay检测趋化、粘附、侵袭应用不同孔径和经过不同处理的聚碳酸酯膜，就可以进行共培养、细胞趋化、细胞迁移、细胞侵袭等多种方面的研究。膜(聚碳酸酯膜polycarbonatemembrane）带有微孔(0.1－12.0µm)细胞:上室，研究下层培养液中的成分对细胞生长、运动等的影响。（1）共培养体系：①细胞不穿过聚碳酸酯膜，常用0.4、3.0µm。

将细胞A种于上室，细胞B种于下室，可以研究细胞B分泌或代谢产生的物质对细胞A的影响。②研究多细胞相互关系血管内皮细胞和中性粒细胞（单核细胞、肿瘤细胞）

（2）趋化性实验

5.0、8.0、12.0µm膜，上室细胞可穿过聚碳酸酯膜进入下室，计数进入下室的细胞量可反映下室成分对上室细胞的趋化能力。

①细胞B对细胞A的趋化作用：将细胞A种于上室，细胞B种于下室，可以研究细胞B分泌或代谢产生的物质对细胞A的趋化作用。

②趋化因子对细胞的趋化作用：将细胞种于上室，下室加入某种趋化因子，可研究该趋化因子对细胞的趋化作用。（3）肿瘤细胞迁移实验常用8.0、12.0µm膜，上室种肿瘤细胞，下室加特定的趋化因子，计数进入下室的细胞量可反映肿瘤细胞的迁移能力。（4）肿瘤细胞侵袭实验

上室种肿瘤细胞，下室加某些特定的趋化因子，聚碳酸酯膜上室侧铺上一层基质胶（模仿体内细胞外基质），细胞欲进入下室，先要分泌基质金属蛋白酶（MMPs）将基质胶降解，方可通过聚碳酸酯膜。计数进入下室的细胞量可反映肿瘤细胞的侵袭能力。

有侵袭能力的细胞才可用于Transwell侵袭实验（实验前检测MMPs的表达，特别是MMP-2的表达。贴壁细胞穿过膜后，可附着在膜的下室侧而不会掉到下室里面去。

细胞染色，可在镜下计数细胞用棉签擦去基质胶和上室内的细胞8.0、12.0µm膜

Tamoxifenextracellularmatrix(ECM)recruitsfewermacrophagesthancontrolECMinatranswellfilterassay.(a)Photomicrographsoftranswellfilters,100×magnification.(b)QuantitationofcellmotilitydemonstratessignificantlyreducedchemotaxistowardstamoxifenECM.*p=0.003.Transwellfilterchemotaxisassayofguineapigbonemarroweosinophils(A)mixedbonemarrowleukocytepopulationplacedintotheupperchamber(B)leukocytesmigratedintothelowerchamberinresponsetoeotaxin(3nmol/L,Eosinophilsshowninred).(C)mixedbonemarrowleukocytepopulationplacedintotheupperTranswellchamber(D)leukocytesmigratedintothelowerchamberinresponsetoeotaxin(E)Totaleosinophilsmigratedinresponsetoeotaxinpresentinthelowerchamber(0to10nmol/L)orupperchamber(3nmol/L;#).(F)Timecourseofeosinophilchemotaxisinducedbyeotaxin(3nmol/L).3.划痕实验4.细胞增殖或增殖抑制实验（人）细胞因子或受体（抗原）+（鼠）特异性单克隆抗体（一抗）+酶标的兔（羊）抗鼠抗体（二抗）（二）免疫学检测法—细胞因子的定量和定位四甲基联苯胺1.2.分泌细胞因子的细胞检测(ELISPOT)Example:HIVVaccineMicewerevaccinatedbyintramuscularinjectionsofplasmidDNAencodingasecretedformoftheHIV-1Gagprotein(pScGag)oranemptyplasmidasacontrol.SomemicereceivedasecondplasmidencodingeitherthenaturalmembraneformofCD40L(pMemCD40L)orthecompany'smultimericsecretedformofCD40L(UltraCD40LorpSP-D-CD40L).Then,spleencellswereanalyzedusinganELISPOTassayforinterferon-gammaproductionwhichvisualizeseachindividualCD8+killerTcellasabrowndot.Asshown,UltraCD40L(pSP-D-CD40L)isasuperioradjuvantforthistypeofantiviralvaccine.Itisnowbeingtestedforthispurposeinmacaquemonkeys组织切片，细胞涂片（抗原）一抗（特异性抗体）洗涤，辣根过氧化物酶标记的二抗3.胞内蛋白或受体的研究（免疫组化，immunohistochemistry)洗涤，辣根过氧化物酶的底物（邻苯二胺）底物转化为产物显微镜下观察直肠腺癌MDR(+)原位杂交图直肠腺癌MDR免疫组化图胃低分化腺癌LMW角蛋白免疫组化图乳腺癌p53免疫荧光图4.(Humantumor)tissuemicroarrays(TMA)Thediagramaboveshowsthebasicmethodtoproducetissuemicroarrays.Anexampleofatissuemicroarrayisshownbelow.Cores(typicallybetween0.6and1mmindiameter)arepunchedoutofpreservednormalandtumourtissuesamplesembeddedinparaffinblocks.Threecoresfromeachtissueareassembledintoanarrayinasecondparaffinblock.Theyarethensectionedwithamicrotomesuchthat50to100arrayscanbemadefromthesametissues.Oncemountedonaglassslide,thesearrayscanbeimmuno-stainedforspecificexpressedtumourmarkersorsubjectedtoFISH(fluorescencein-situhybridization)analysis.fulltissuemicroarrayimageIntheexamplesshown,thearraysarefirstimmunoperoxidasestained(yellow-brown)withaspecificantibodyagainstatumourproteinexpressedinthecells,thencounterstainedwithhematoxylin(blue-red)torevealthecellnuclei.Thetumourtissuecanbedifferentiatedfromhealthytissuebythepreponderanceofbrownstaining.magnifiedsectionoftissuemicroarray5.免疫印迹法(Westernblotting)

MstainMarkerSampleSDS硝酸纤维膜聚丙烯酰胺凝胶电泳+-硝酸纤维膜一抗（特异性抗体）洗涤，辣根过氧化物酶标记的二抗底物WesternblotPAGE(X2)MMstainNo-stain1-3h_+Transferassemble'brillo'padsWhatman#1filterpaperPAGEgelnitrocellulosepositiveelectrodenegativeelectrodestainImmersedinasolutionoftheantibodywashingImmersedinasolutionofthesecondantibodywashing(IgG-HRP)MM6.免疫共沉淀（Co-Immunoprecipitation,Co-IP）6.免疫共沉淀（Co-Immunoprecipitation,Co-IP）7.基于流式细胞仪的液相蛋白定量技术CytometricBeadArray（CBA）微量样本多指标流式蛋白定量技术Fig.3Concentrationsofcytokinesmeasuredinnonallergicvs.allergicdonortears.Cytometricbeadarray:amultiplexedassayplatformwithapplicationsinvariousareasofbiologyCytokinemeasurementintearsamplesTearsareinvolvedinantimicrobialdefense,woundhealing,andinflammatoryresponses.Variousoculardiseasestatescauseelevationofinflammatorycytokinelevelsintears.However,characterizationoftearcytokineshasbeenlimitedbythesmallamountsattainablefortesting.Tearsamples(5~10μl)fromnonallergicandallergicdonorswereincubatedfor20hatroomtemperaturewithamixtureofcaptureantibodybeadsanddetectionantibodies.Thebeadswerewashedtoremoveunbounddetectionantibodyandwereanalyzedusingflowcytometry.Allsixcytokinesweredetectableinbothnonallergicandallergictears.TearsfromallergicdonorscontainedsignificantlylessIL-10thantearsfromnonallergicpatients.(Fig.3).ThereducedlevelofIL-10inthetearsofallergicdonorscouldbeanimportantattributeofthepathophysiologyofocularallergicinflammation.（三）分子生物学检测法—检测细胞因子的mRNA表达1.RT-PCR检测基因表达不能提供因子的浓度和活性RT-PCR的原理

2.实时定量PCR技术的原理及应用(RealtimeQuantitativePCR)实时荧光定量PCR定义

在PCR反应体系中加入荧光基团，利用荧光信号累积实时监测整个PCR进程，最后通过标准曲线对未知模板进行定量分析的方法实时荧光定量PCR原理常规PCR技术：

对PCR扩增反应的终点产物进行定量和定性分析无法对起始模板准确定量，无法对扩增反应实时检测实时定量PCR技术：利用荧光信号的变化实时检测PCR扩增反应中每一个循环扩增产物量的变化，通过Ct值和标准曲线的分析对起始模板进行定量分析实时荧光定量PCR方法SYBRGreen法SYBRGreen工作机理SYBRGreen能结合到双链DNA的小沟部位SYBRGreen只有和双链DNA结合后才发荧光变性时，DNA双链分开，无荧光复性和延伸时，形成双链DNA，SYBRGreen发荧光，在此阶段采集荧光信号。SYBRGreen实时定量PCR原理SYBRGreenI5’3’5’3’SGNoEmissionSGSGSGExcitationSGSGSGSGSGSGSG5’3’5’3’EmissionExcitationSetup荧光信号经过滤光片再聚焦到CCD上实时荧光定量PCR原理

扩增曲线扩增曲线图：横坐标：扩增循环数纵坐标：荧光强度，每个循环进行一次荧光信号的收集荧光基团荧光检测元件实时荧光定量PCR原理Ct值Ct值的定义：

PCR扩增过程中，扩增产物的荧光信号达到设定的阈值时所经过的扩增循环次数C(t)value实时荧光定量PCR原理

定量原理理想的PCR反应：

X=X0*2n非理想的PCR反应：

X=X0(1+Ex)nn：扩增反应的循环次数