免疫-楊勇軍 細(xì)胞死亡機(jī)制

Celldeath（細(xì)胞死亡）ReversiblecellinjuryFunctionalandmorphologicchangesarereversibleifthedamagingstimulusisremoved.Thefeaturesare:decreasedoxidativephosphorylation,ATPdepletionandcellularswelling.Reversibledamage–cellularswellingHowmuchcanacellswell?IrreversibleinjuryandcelldeathWithcontinuingdamage,injurybecomesirreversible.Cellsundergomorphologicchangesrecognisableascelldeath.MechanismsofcellularinjuryBymorphologyNecrosisTypeICD:apoptosis,pyroptosis,mitoticcatastropheTypeIICD:autophagyTypeIIICD:necroptosis,paraptosis,oncosisBymechanismCaspase-dependentcelldeath(CDCD):typeICDCaspase-independentcelldeath(CICD):necrosis,typeII&IIICDTypesofCellDeath“gene–directedcellularself-destruction”orprogrammedcelldeath.Apoptosis細(xì)胞凋亡是一個(gè)主動(dòng)的由基因決定的自動(dòng)結(jié)束生命的過程，所以也常常被稱為細(xì)胞編程死亡（programmedcelldeath,PCD）。生物學(xué)意義：細(xì)胞凋亡對(duì)于多細(xì)胞生物個(gè)體發(fā)育的正常進(jìn)行，自穩(wěn)平衡的保持以及抵御外界各種因素的干擾方面都起非常關(guān)鍵的作用.ApoptoticBCells"Webbed"tissuebetweenthedigitsofdevelopinghumanembryosisremovedbyapoptosisTadpoletailremovedbyapoptosisduringdevelopmentNeuronalcelldeathbyapoptosisisfundamentalforCNSdevelopmentEssentialforproperdevelopmentofthemulticellularorganismCellsoftheintestinalwalldiebyapoptosistobereplacedbynewcellsSkincells(keratinocytes)undergoapoptosisandmigratetothesurfacewheretheyformtheprotectiveouterlayerofskin

Essentialfortheproperfunctioningofthematureorganism

EssentialfortheremovalofcellsthatthreatenhomeostasisVirus-infectedcells,cancer,CTLCellswhoseDNAhasbeendamagedbyUVlight,exposuretoradiation,chemotherapyAutoreactiveTcellswiththepotentialtoattack"self"areremovedbyapoptosisAttheterminationofanimmuneresponsewhentheyarenolongerneededSydneyBrenner,H.RobertHorvitz,JohnESulstonApoptosisinC.elegans1.形態(tài)學(xué)特征●細(xì)胞體積變小，細(xì)胞質(zhì)濃縮?！癜そY(jié)構(gòu)完整，有泡狀突起。●細(xì)胞核染色質(zhì)濃縮，聚集核膜周圍?！窦?xì)胞膜內(nèi)陷形成凋亡小體

(Apoptoticbodies)?！駸o內(nèi)容物外溢，因而不引起周圍的炎癥反應(yīng)。2.細(xì)胞凋亡的生化特征染色質(zhì)DNA斷裂,形成180~200bp整數(shù)倍的DNA片段；細(xì)胞內(nèi)的酶被激活；細(xì)胞內(nèi)活性氧分子增多、胞漿鈣離子升高。膜內(nèi)側(cè)磷脂酰絲氨酸外翻到膜表面線粒體膜電位消失，膜通透性變大，細(xì)胞色素C被釋放。Early:1.Decreasedcellsize(celldehydration)2.Alteredcellmembrane3.LargeDNAstrandbreak4.IncreaseincellularcalciumlevelsIntermediate:1.DNAcleavageinto180~200bpfragments,whichgivethecharacteristic“l(fā)addering”onaDNAgel.2.Furtherdecreaseincellsize3.Alterationsinplasmamembranesymmetry4.DecreasedpHLate:1.Lossofmembranefunction2.Apoptoticbodies.Threestages細(xì)胞膜泡狀化（blebbing）Caspase3剪切膜蛋白，干擾膜蛋白的代謝。ApoptosisindevelopmentofCaenorhabditis

elegans

ThelifecycleofC.elegansfromeggtosexualmaturity(andneweggs)isabout3days.Theadulthermaphroditeconsistsofexactly959somaticcellsofpreciselydeterminedlineageandfunction.Individualcellsarenamedandtheirrelationshipstotheirneighboursareknown.Overallthe959cellsofadultC.elegansarisefrom1090originalcells;exactly131cellsundergoprogrammedcelldeathinthewildtypeworm.Ofthe1090,302areneurons,andmanyoftheprogrammeddeathsalsolieintheneuronallineage.細(xì)胞凋亡機(jī)制GeneticMechanismofApoptosis:TheapoptoticsysteminC.elegans.Meieret.alNatReviews,Vol407,2000.

MolecularnatureofthePCDcamefromgeneticstudiesdoneonC.elegans.131cellsoutof1090cellsundergoPCD:geneticscreensofmutantsidentifiedEgl-1,CED3,CED4andCED9tobeinvolved.Egl-1,CED3,CED4aredeathpromoterssincetheirlossoffunctionresultsinsurvivalofall131doomedcells.

CED9isdeathinhibitorsinceitslossoffunctioncausesembryoniclethalitybymassiveectopiccelldeaths.KauffmannandVaux.Oncogene,22,2003

Fundamentalcomponentsofapoptoticpathwaysareconservedacrossthespecies.

Ced-9issimilartohumanoncogene:Bcl2.

Ced-3hasamammaliancounterpart,originallyknownasICE(Interleukin1?ConvertingEnzyme),nowtermedCaspase1.

Ced-4actsasanadapterforcaspaseactivation;themammaliancounterpartApoptosisactivatingfactorApaf-1.

MolecularIdentitiesofapoptoticgenes細(xì)胞凋亡的信號(hào)通路（一）內(nèi)源性通路（線粒體途徑）（二）外源性通路（死亡受體途徑）:

Fas,TNF,Trail.（三）共同終末：caspase激活，啟動(dòng)細(xì)胞凋亡進(jìn)程Mitochondrialpathway線粒體不僅是細(xì)胞能量代謝的中心，而且在細(xì)胞內(nèi)源性的死亡信號(hào)所誘導(dǎo)的細(xì)胞凋亡中處于中心位置。細(xì)胞內(nèi)部的死亡信號(hào)源自各種因素，如DNA損傷、氧化應(yīng)激、X-射線、化療藥物、營養(yǎng)缺乏等。因而，線粒體所介導(dǎo)的凋亡途徑又稱為細(xì)胞凋亡的內(nèi)源途徑。（一）內(nèi)源性通路：（Intrinsicpathway）線粒體介導(dǎo)凋亡的大致過程：

損傷因素→線粒體跨膜電位（△ψ）的消失和線粒體滲透轉(zhuǎn)運(yùn)孔（PTP）的開放→細(xì)胞色素C從線粒體中的釋放和Caspase-9的激活。（二）外源性通路（Extrinsicpathway）TheDeathReceptorFamily

CellsurfacecytokinereceptorsbelongingtoTNF/NGFreceptorsuperfamily.ReceptorsareTypeItransmembraneproteins:intracellularCterminaltail,membranespanningregion,anextracellularligandbindingdomain.Significanthomologyin60-80aacytopl.sequence–Deathdomain(DD).Deathreceptorsareactivatedbytheirnaturalligands:groupofcytokinesbelongingtoTNFfamily.DeathReceptorSignaling

Ligandbindingtothedeathreceptorsleadstooligomerizationofthesereceptors.RecruitmentofadaptormoleculesthroughtheirDeathDomains(DD)Thisprotein–proteininteractionisrestrictede.gFas,DR4,DR5recruitsFADDwhereasTNFR1recruitsTRADD.AdaptormoleculesrecruitinitiatorcaspasesthroughinteractionoftheirDeatheffectorDomain(DED)andCARDdomainincaspases.TheresultingcomplexiscalledDeathinducingSignaling(DISC)complex.1.Fas/FasLpathway1）Fas/FasL蛋白：Fas又稱CD95或凋亡蛋白-1(Apo-1)，分子量約為43kD，成熟蛋白長(zhǎng)319氨基酸。胞膜外區(qū)有3個(gè)富含半胱氨酸的結(jié)構(gòu)域(cysteine-richdomain,CRD)，其中有2個(gè)N-糖基化位點(diǎn)。胞漿部分含有70個(gè)左右DD(deathdomain)區(qū)。FasL分子量約40kD，成熟蛋白長(zhǎng)281個(gè)氨基酸。2）分布：Fas表達(dá)比較廣泛，胸腺、心臟、肝、肺、腎和卵巢等都有表達(dá)。與Fas分布廣泛性不同的是，F(xiàn)asL通常只出現(xiàn)于活化的T細(xì)胞和NK細(xì)胞。3）Fas誘導(dǎo)凋亡的途徑：Fas-FADD-Caspase-8FasL與Fas結(jié)合后，F(xiàn)as三聚體化。通過DD區(qū)的蛋白質(zhì)之間的相互作用，F(xiàn)as與FADD(fasassociateddeathdomain)結(jié)合，形成DISC(deathinducingsignalingcomplex)。通過FADD的DED(death

effectordomain)區(qū)，Procaspase-8被募集在DISC上。2.TNFpathwayTNF(Tumornecrosisfactor):TNF由活化的單核巨噬細(xì)胞產(chǎn)生，亦稱惡液質(zhì)素；TNF由活化的T細(xì)胞產(chǎn)生，亦稱淋巴毒素。兩種TNF的分子量為51kD，結(jié)構(gòu)為同源三聚體，TNF-α和TNF-β都可以分別與TNFRⅠ或TNFRⅡ結(jié)合，并且與這兩型受體的結(jié)合基本上沒有選擇性。TNFR特點(diǎn)TNFRⅠ又稱為TNFR55，胞膜外區(qū)有4個(gè)CRD，其中有3個(gè)N-糖基化位點(diǎn)。胞漿區(qū)有3個(gè)蛋白激酶C(PKC)作用位點(diǎn)，1個(gè)蛋白酪氨酸激酶(PTK)的作用位點(diǎn)。TNFRⅡ又稱為TNFR75，胞膜外區(qū)也有4個(gè)CRD，其中有2個(gè)N-糖基化位點(diǎn)。跨膜區(qū)有1個(gè)PKC作用位點(diǎn)。TNFRⅠ與TNFRⅡ在胞膜外區(qū)有28%的同源性，在胞漿區(qū)沒有同源性。TNFRⅠ在胞漿區(qū)有一個(gè)約80個(gè)氨基酸的DD區(qū)，TNFRⅡ沒有同樣的結(jié)構(gòu)域，但其C端的78個(gè)氨基酸殘基可以結(jié)合信號(hào)轉(zhuǎn)導(dǎo)蛋白TRAF2(Tumornecrosisfactorreceptor-associatedfactor-2)等胞漿信號(hào)蛋白，在信號(hào)轉(zhuǎn)導(dǎo)中發(fā)揮重要作用。TNF誘導(dǎo)的凋亡途徑TNFRⅠ-TRADD(TNFreceptor-associateddeathdomainprotein)-FADD-Caspase-8TNF與TNFRⅠ結(jié)合后，TNFRⅠ三聚體化。通過DD區(qū)，TRADD不但可以結(jié)合TNFRⅠ的胞漿區(qū)，也可以結(jié)合FADD的C-端部分。通過FADD的DED區(qū)，Procaspase-8被募集在DISC上。聚集的Procaspase-8或10，通過自身或相互切割產(chǎn)生成熟的Caspase-8或10。

TNFRⅠ活化轉(zhuǎn)錄因子NF-κB的信號(hào)也是通過TRADD介導(dǎo)的，TRADD在通過C端部分的DD區(qū)與TNFRⅠ胞漿區(qū)結(jié)合后，可以與另一種信號(hào)轉(zhuǎn)導(dǎo)蛋白TRAF2(Tumornecrosisfactorreceptor-associatedfactor-2)結(jié)合，TRAF2再通過NIK→IKK→IκB通路引起NF-κB的活化，使細(xì)胞存活。TNF誘導(dǎo)的細(xì)胞存活途徑3.TRAILpathway●TRAIL又稱凋亡蛋白2配體（Apo2ligand）;●由281個(gè)氨基酸組成，分子量32kD;●其胞膜外C端區(qū)域與FasL和TNFα的同源性分別為28%及23%;●TRAIL受體：DR4又稱Apo-2或TRAILR1，胞膜外區(qū)有2個(gè)CRD，在C端的胞漿區(qū)有DD區(qū)，與TNFRI相應(yīng)的結(jié)構(gòu)域有30%的同源性。

TRAILR—DR5（TRAILR2），其胞膜外區(qū)也有2個(gè)CRD，胞漿區(qū)有DD區(qū)?！裨谡：湍[瘤細(xì)胞中，DR4和DR5分布均非常廣泛●最顯著的特征就是能夠選擇性殺傷腫瘤細(xì)胞而對(duì)大多數(shù)正常細(xì)胞沒有明顯毒性。DCR1和DCR2的抗凋亡作用●DCR1（Deathdecoyreceptor1）也被稱為TRID(TRAILreceptorwithoutanintracellulardomain)。胞膜外區(qū)也有2個(gè)CRD，與DR4和DR5分別有69%和52%的同源性。但缺少DD區(qū)?！馜CR2的胞膜外區(qū)與其他TRAILR相似，但它胞漿部分的DD區(qū)不完整?！穹植迹篋CR1和DCR2只表達(dá)于正常組織，在腫瘤細(xì)胞系和轉(zhuǎn)化細(xì)胞不表達(dá)?！馜CR1和DCR2的抗凋亡作用：DR4、DR5、DCR1和DCR2都可以與TRAIL結(jié)合。TRAIL與DR4和DR5的結(jié)合可以引起細(xì)胞凋亡，而DCR1和DCR2由于沒有胞漿部分或不完整，不能向胞漿中轉(zhuǎn)導(dǎo)凋亡信號(hào)，所以DCR1和DCR2與TRAIL結(jié)合后，可以保護(hù)細(xì)胞不發(fā)生凋亡。FADD?Caspase-8Caspase-10（三）共同終末：caspasecascadeWhatiscaspases●Caspase：cysteine-containingaspartate-specificproteases●Agroupofcysteineproteaseswithacrucialcysteineresiduethatcancleaveotherproteins,afteranasparticacidresidue,aspecificitywhichisunusualamongproteases.●

Caspasesareessentialincellsforapoptosis.Caspase的種類已鑒定的caspase家族成員有14種：人類12種(無caspase-11、-12)小鼠9種(無caspase-4、-5、-9、-10、-13)不參加caspase編序的有：

C.elegans的7種蛋白酶果蠅的5種蛋白酶。Caspase家族特征Caspase通常以酶原（Procaspase）的形式存在，相對(duì)分子質(zhì)量為29-49kD。N末端具有一個(gè)原結(jié)構(gòu)域（Prodomain）C端包含約20kD的大亞基和約10kD的小亞基C端同源區(qū)存在半胱氨酸激活位點(diǎn)，此激活位點(diǎn)結(jié)構(gòu)域?yàn)镼ACR/QG。根據(jù)在細(xì)胞凋亡中的作用，Caspases分為兩大類：?jiǎn)⑹糃aspase(Initiator):包括-2，-8,-9,-10等，對(duì)細(xì)胞凋亡的刺激信號(hào)作出反應(yīng)，啟動(dòng)細(xì)胞的凋亡過程；效應(yīng)Caspase(Effector):包括-3，-6，-7等，是在細(xì)胞凋亡過程中的具體執(zhí)行者，完成對(duì)特定蛋白底物的水解。Caspase激活●去除N端原結(jié)構(gòu)域，在大小亞基中進(jìn)行切割；●大小亞基形成異源二聚體，進(jìn)而形成四聚體。Caspase的效應(yīng)機(jī)制激活的Caspase可以水解約100個(gè)底物，包括細(xì)胞調(diào)節(jié)、細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)、DNA修復(fù)、組織平衡、細(xì)胞存活等環(huán)節(jié)中重要的蛋白，從而使細(xì)胞表現(xiàn)為凋亡特有的形態(tài)學(xué)及生化特征：細(xì)胞皺縮、斷裂，染色質(zhì)聚集，DNA降解，以及隨后的凋亡細(xì)胞被吞噬細(xì)胞迅速地清除等。三、重要的凋亡控制基因BCl-2家族p53IAP家族1.BCl-2家族●

Bcl-2是調(diào)節(jié)細(xì)胞凋亡途徑中的一個(gè)重要基因?！窈髞碛职l(fā)現(xiàn)許多基因結(jié)構(gòu)與Bcl-2相似，稱之為Bcl-2基因家族蛋白產(chǎn)物。●根據(jù)它們對(duì)細(xì)胞凋亡作用結(jié)果的不同，可將Bcl-2家族成員分為抑制凋亡和促進(jìn)凋亡兩大類。2.P53參與DNA損傷而誘發(fā)的細(xì)胞凋亡●使細(xì)胞在G1期停滯：當(dāng)DNA由于各種原因損傷后，p53首先誘導(dǎo)細(xì)胞進(jìn)入G1期，抑制細(xì)胞增殖，直至損傷的DNA修復(fù)?！褚坏〥NA不能被修復(fù)，p53就會(huì)活化那些誘導(dǎo)細(xì)胞凋亡的基因轉(zhuǎn)錄，使細(xì)胞發(fā)生凋亡。3.IAPIAP(inhibitorofapoptosisprotein)為一組具有抑制凋亡作用的蛋白質(zhì)，首先是從桿狀病毒（Baculovirus）基因組克隆到。所有的IAP都含有對(duì)抗凋亡作用非常重要的70氨基酸的BIR重復(fù)序列（Baculovirus

IAPRepeat）。在C末端還擁有一個(gè)高度保守的RING序列，因而它們具有泛素（Ubiquitin）E3的活性。HumancellularIAPs(BIRPs)IAP抗凋亡的機(jī)制通過BIR序列與Caspase蛋白質(zhì)-蛋白質(zhì)作用，IAP可以直接抑制Caspase-3、7和9的作用。通過RING序列，IAP可以催化Caspase-3和-7泛素化，導(dǎo)致這些Caspase被proteasome降解。TechniquestodetectApoptosis1)VitalDyeexclusionassay:

Trypanblue,propidiumiodide:donotstaintheviablecells

Fluorescein

diacetate,Calcein-AM:staintheviablecells.2)MeasurementofcytosolicLeakage:basedonthefactthatviablecellshaveintactcellularcomponents.

LactateDehydrogenase

Pyruvate+NADHLactate+NAD+NADHNADHexhibitsfluorescenceatanexcitationwavelengthof360nmwithemissionat450nm.VelocityofdecreaseofEx360nm/Em450nmindicatesconversionofNADHtoNADandhenceactivityofLDH.3)Clonogenicassay:

Abilityofcellstodivideandformcoloniesiscalledclonogenicactivity.However,integrityofplmembisnotcompromisedtilllatestageandhencenotveryuseful;

DeterminationofCellViabilityTest:Theeasiestwaytomeasurecelldeathisbymeasuringlactatedehydrogenase(LDH),astablecytoplasmicenzymewhichispresentinallcellsbutonlyreleasedwhentheplasmamembraneisdamaged.Cell-mediatedcytotoxictywasmeasuredwiththeLDHCytotoxicityDetectionKit.MousespleencellswerestimulatedinvitroandcytotoxicTlymphocytes(CTLs)wereisolated.10,000testcells/wellwereincubatedwiththeeffector

CTLs,andthencelldeathwasmeasuredusingtheLDHassay.ClonogenicassayAlterationsinPlasmaMembrane:

Innormalcells,phosphoidylcholineandsphingomyelinareonexternalleafand

phosphotidylethnolamineandphosphatidylserine(PS)areintheinnerleaflet.Redistributionofphospholipidsintheplasmamemb.isanearlyapoptoticchange.

AnnexinVconjugatedwithflourophorelikeFITCcanbindtoexposedtoPSinCadependentmanner.Canbeviewedmicroscopicallyorbyflowcytometry.AnnexinVstainingAlterationsinCytosol:caspaseactivation

Activationofcaspases:hallmarkofapoptosis,canbemeasured.WesternblotforPARP,smallsubunitsofcaspase3,7,8and9.Biochemicalanalysisofcaspaseactivity:

Tetrapeptidesequence(recognitionsiteofcaspase)conjugatedwithreportgrouplikep-nitroanilide(pNA)7-amino-4-methylcoumarin(AMC)7-amino-4–triflouromethylcoumarin(AFC)Calorimetric/flourimetricmeasurements.

Immunohistochemica

Immunostaining/flourescentsubstratesintissues/cells.l/immunoflorescentstainingonparaffinsections.Cellpermeableflourescentsubstrates:PhiphiLux(Oncogeneresearchproducts)Bcl2familyProteins:

Westernblotwithantibodiesspecifictoindividualmembers.Ifsubcellular

localisationisnotrelevant:cellscanbelysedinnon-ionicdetergent.Ifsubcellularisneeded,cellularcomponentsaresubfractionatedtoobtainmitochondrialandcytosolicfractions.TodetermineBaxorBak

oligomers,crosslinkingagentsareaddede.g

disuccinimidyl

suberate(DSS),

Bis(Sulphosuccinimidyl)suberate(BS3)tothemitochondrialfractionsuspendedinisotonicbuffer.Thecrosslinkerisquenchedwith1MTris-HClph7.5.Membranesarethenlysedinradioimmunolprecipitationassay(RIPA)bufferandclearedbycentrifugationat12000g:analysisbySDSandWesternBlot.

OligomerisationisvisualisedusingWesternblotasahighmolecularweightspecies.

Cytcreleaseisthemostcommonparameterofactivemitochondrialpathway.

Subcellularfractionationtoyieldmitochondrialfractionwhichissuspendedinisotonicbufferwithenergisingagents.

WesternBlotofbothsupernatantandpelletwithcytcantibody:cytcinsupandreductioninthepelletindicatecytcrelease.ELISA:SupernatantispreparedforELISAfordetectionofCytc.ImmunostainingcellsundergoingapoptosisarewashedandfixedincubatedwithanticytcAbWashandincubationwithsecAbtaggedwithflourophore.Diffusedcytoplasmicstainingindicatescytcrelease

MitochondrialreleaseofCytCMitochondrialChanges:MitochondrialTransmembranePotentials:

Mitochondriatransmembranepotentialisafunctionalparameterduringapoptosis.Canbedeterminedbylipophilic

cations:accumulationispotentialdependent.Commonlyusedare:Rhodamine123(Rh123),DiOC6,Tetramethylrhodamine

methylester(TMRM),JC-1.Probescanbedirectlyaddedtoculturedcellsorisolatedmitochondria,incubatedfor15minandharvestedtobeanalyzedbyfloworfluorescentmicroscopy.

ChangesintheNucleus:NuclearCondensationandFragmentation:

Nuclearcondensationandfragmentationcanbevisualisedbystainingwithfluorescentdyese.gHoechst(bisbenzimide)andDAPI(4’6’diamidino2-phenylindole,dilactate).Intensityofstaininginthenucleusisproportionaltotheextentofapoptosisduetoincreasedpermeabilityofthedyes.DNAcontentstainingbyPropidiumIodide::

DegradationofnuclearDNAresultsindecreaseinDNAcontentorhypoploidy.CellsaresuspendedinicecoldPBS,fixedwithcoldethanol.CellsarepermeabilisedwithTritonx100,stainedwithPIandanalysedwithflow.However,necroticandothercellulardebriscanalsogetincluded.DNA細(xì)胞周期分析細(xì)胞周期G2MG0G1s

200

400

600

8001000G0G1sG2MDNA分析DNA含量細(xì)胞數(shù)量2N4NDNA降解分析CAD核酸酶（Caspases-activatedDNase）可以造成凋亡時(shí)DNA的片段化。在正常情況下，CAD不顯示活性，因?yàn)镃AD以一種無活性的復(fù)合物形式（CAD-ICAD）存在。細(xì)胞凋亡時(shí)，Caspases水解ICAD，CAD就會(huì)處于活性狀態(tài)并最終使DNA片段化。瓊脂糖凝膠電泳DNAFragmentation:

CleavageofDNAatnucleosomalsitesresultsin180-200bpfragmentswhichappearsasDNAladder.Quantitativemeasurement:amountoffragmentedDNAisproportionaltofrequencyofapoptosis.DetectionofDNAbreaksbyTUNELassay:(Terminaldeoxynucleotidyl

transferase(TDT)mediateddUTP

nickendlabeling)

BasedontheprinciplethatTdTmediatesincorporationofbiotinylated

dUTPinto3’OHendsoffragmentedDNA.CellsarepermeabilisedusingTritonX100orProteinaseKincaseofformalinfixedtissuesections.Cells/TissuesectionsaretheincubatdwithTdTanddUTP-biotin.Afterwash,cells/TissuescanbeincubatedeitherwithFITCconjugatedavidinfordetectionunderfluoroscentmicroscopeoravidin-HRPconjugatecanbeaddedfollowedbyDAB.細(xì)胞自噬（Autophagy）1962年Ashford等在胰高血糖素處理的小鼠肝細(xì)胞中觀察到autophagy1963年,DeDuve首次提出細(xì)胞自噬的生物學(xué)概念:細(xì)胞在缺乏營養(yǎng)和能量供應(yīng)時(shí)，

部分細(xì)胞質(zhì)與細(xì)胞器被包裹進(jìn)一種特異性的雙層膜或者多層膜結(jié)構(gòu)的自噬體(autophagosome)中，形成的自噬體再與溶酶體(Lysosome)融合形成自噬溶酶體(autolysosome)，胞質(zhì)和細(xì)胞器成分在這里被降解為核苷酸、氨基酸、游離脂肪酸等小分子物質(zhì)，這些小分子物質(zhì)可以被重新利用合成大分子或者合成ATP。Autophagyautophagyisaprocessbywhichcellsundergopartialautodigestionthatprolongssurvivalforashorttimeunderstarvationconditions.Itprovidesnutrientsthatarenecessarytomaintaincellviability.autophagyisalsoinvolvedinthekillingofbacteriathatareingestedbycells.細(xì)胞生存的一種機(jī)制,在很多生理過程如清除損傷、衰老細(xì)胞器以及冗余蛋白上發(fā)揮著重要作用Autophagy細(xì)胞利用溶酶體降解自身受損的細(xì)胞器和大分子物質(zhì)的過程，是真核細(xì)胞特有的生命現(xiàn)象細(xì)胞內(nèi)物質(zhì)的兩種降解途徑：蛋白酶體系統(tǒng)：降解胞內(nèi)的短壽命蛋白自噬作用：長(zhǎng)壽命蛋白和一些細(xì)胞器的降解利用theproteasomebreaksdownubiquitinatedproteins,butitmaynotrecognizemisfoldedproteinsandproteinaggregatesVesiclecontaining

twodamaged

organelles1mMitochondrion

fragmentPeroxisome

fragmentPeroxisomeVesicleMitochondrionLysosomeDigestion(b)AutophagyAutophagyisactivatedbyChangingofenvironmentalconditions:starvationconditionsassociatedwithdeficiencyofnutrientssuchasaminoacids;hypoxicconditions;hightemperaturesCellularremolding:duringdevelopmentanddifferentiation（誘導(dǎo)因素：營養(yǎng)和能量缺乏、氧化應(yīng)激、感染、蛋白質(zhì)大量聚集）小自噬

2大自噬（一般）

1

3分子伴侶介導(dǎo)的自噬（CMA）：由內(nèi)質(zhì)網(wǎng)來源的膜包繞待降解物形成自噬體，然后與溶酶體融合并降解其內(nèi)容物；分子伴侶介導(dǎo)的自噬（CMA）：溶酶體的膜直接包裹長(zhǎng)壽命蛋白等，并在溶酶體內(nèi)降解；胞質(zhì)內(nèi)蛋白結(jié)合到分子伴侶后被轉(zhuǎn)運(yùn)到溶酶體腔中，然后被溶酶體酶消化。CMA的底物是可溶的蛋白質(zhì)分子，在清除蛋白質(zhì)時(shí)有選擇性，而前兩者無明顯的選擇性。

根據(jù)細(xì)胞物質(zhì)運(yùn)到溶酶體內(nèi)的途徑不同細(xì)胞自噬過程自噬體膜：來源：粗面內(nèi)質(zhì)網(wǎng)的非核糖體區(qū)域高爾基體一種新合成的結(jié)構(gòu)被降解物：部分胞漿細(xì)胞內(nèi)需降解的細(xì)胞器(線粒體)細(xì)胞內(nèi)需降解的蛋白質(zhì)細(xì)胞自噬過程饑餓、氧化應(yīng)激損傷→自噬體膜脫落，形成環(huán)狀分隔膜，包繞在被降解物周圍分隔膜逐漸延伸，將要被降解的胞漿成分完全包繞形成自噬體（autophagosome）自噬體通過細(xì)胞骨架微管系統(tǒng)運(yùn)輸至溶酶體，與之融合形成自噬溶酶體(autolysosome),并降解其內(nèi)成分，自噬體膜脫落再循環(huán)利用自噬的過程自噬的過程自噬的功能對(duì)外源性刺激(包括營養(yǎng)缺乏、細(xì)胞密度負(fù)荷、低氧、氧化應(yīng)激、感染等)的適應(yīng)性反應(yīng)：降解產(chǎn)物氨基酸、核苷酸、游離脂肪酸等可供物質(zhì)能量循環(huán)細(xì)胞保持穩(wěn)定狀態(tài)的管家機(jī)制：調(diào)控長(zhǎng)壽命蛋白、過氧化物體、線粒體和內(nèi)質(zhì)網(wǎng)的更新參與一定的組織特異性融合

一種防御機(jī)制:清除胞質(zhì)內(nèi)受損的細(xì)胞器、代謝產(chǎn)物,進(jìn)行亞細(xì)胞水平上的重構(gòu),保護(hù)受損的細(xì)胞；作為一種細(xì)胞死亡程序誘導(dǎo)細(xì)胞主動(dòng)性死亡細(xì)胞自噬的分子機(jī)制參與自噬體形成的兩個(gè)泛素樣蛋白系統(tǒng)：分別由Atg3、Atg5、Atg7、Atg10、Atg12和LC3參與組成Atg12首先由E1酶Atg7活化，之后轉(zhuǎn)運(yùn)至E2酶Atg10，最后與Atg5結(jié)合,形成自噬體前體(autophagosomalprecursor)；LC3-Ⅰ也被Atg7活化，轉(zhuǎn)運(yùn)至第二種E2酶Atg3，并被修飾成膜結(jié)合形式LC3-Ⅱ。LC3-Ⅱ定位于前自噬體和自噬體——自噬體的標(biāo)志分子（LC3是酵母細(xì)胞自噬相關(guān)基因Atg8的類似物）細(xì)胞自噬的分子機(jī)制Ⅲ型磷脂酰肌醇三磷酸激酶（ClassⅢPI3K）PI3kinasetypeIII,whichincludesAtg6initscomplex,promotesthenucleationofautophagicvesicles.自噬的生理學(xué)意義自噬在生理過程的作用：參與發(fā)育和分化過程中機(jī)體的重新構(gòu)建(remomding)營養(yǎng)缺乏時(shí)產(chǎn)生氨基酸清除不需要的、損傷的細(xì)胞器與分子自噬的生理和病理意義廣泛存在于正常的生理過程中：如清除細(xì)胞廢