益生菌衍生的可溶性因子對于宿主防御的調(diào)節(jié)機制：對消化系統(tǒng)的健康影響

FangYan,M.D.,Ph.D.DepartmentofPediatricsDivisionofGastroenterology,HepatologyandNutritionVanderbiltUniversitySchoolofMedicineNashville,TNSupport:NationalInstituteofDiabetesandDigestiveDiseasesandKidneyDiseases,Crohn’sandColitisFoundationofAmerica,VanderbiltDigestiveDiseaseResearchCenter,theNestleNutritionGrant,andAtticus-BrownFamilyTrustProbiotic-derivedsolublefactorsregulatehostdefensemechanisms:impactondigestivehealth糖尿病、消化系統(tǒng)疾病和腎病國家研究所、美國克洛病和結(jié)腸炎基金會、范德比特消化系統(tǒng)疾病研究中心、雀巢營養(yǎng)公司的授權(quán)、布朗家族信托益生菌衍生的可溶性因子對于宿主防御的調(diào)節(jié)機制：對消化系統(tǒng)的健康影響范德比特大學(xué)醫(yī)學(xué)院腸道學(xué)、肝膽學(xué)和營養(yǎng)學(xué)部門兒科田納西州納什維爾Researchprojects:

1.Preventionandtreatmentofintestinalinflammation

-Probiotic(LactobacillusGG)-derivedsolubleproteins

-Berberine(analkaloidisolatedfromplants)2.TherolesofEGFandTNFsignalingpathwaysinregulationofinjuryand repairmechanismsinintestinalinflammationandcarcinogenesis

3.ThemechanismsofH.pylori-inducedgastricinflammationandcancer development

預(yù)防和治療腸道炎癥-LGG的分泌蛋白-黃連素（一種從植物中分離的生物堿）-可治療腹瀉

EGF和TNF的信號通路在腸道炎癥及癌變中對損傷和修復(fù)機制的調(diào)節(jié)作用。幽門螺桿菌誘導(dǎo)胃炎癥和癌變的機制

Intestinalepithelialcellhomeostasisandfunctions

腸上皮細(xì)胞的動態(tài)平衡和功能

Digestionandabsorption

WaterandelectrolytetransportProtectivedefensemechanisms

Barrierfunction(tightjunctions)

Anti-bacterialsubstancesInnateandadaptiveimmunity

腸細(xì)胞保護(hù)防御機制屏障功能腺管小腸絨毛杯狀細(xì)胞潘氏細(xì)胞內(nèi)分泌細(xì)胞干細(xì)胞先天免疫后天免疫抗菌物質(zhì)Thegastrointestinalmicrobiota1014GIbacteria1013cellsinthebody1012skinbacteriaOralcavity200species

StomachH.PyloriSmallintestine108bacteria/mlColon1010-1011bacteria/g400-500speciesBacteriodesEubacteriumBifidobacteriumRuminococcusBacillusClostridiumLactobacillusEnterococcusEnterobacter桿菌屬真菌屬雙歧桿菌屬瘤胃球菌屬芽孢桿菌屬梭菌屬乳桿菌屬腸球菌屬腸桿菌屬胃腸道細(xì)菌幽門螺桿菌Commensalbacteriainthegastrointestinaltract胃腸道中的共生細(xì)菌Beneficialeffects:

Promoteintestinaldevelopment.

Benefithosthealthbyenhancingpolysaccharidedigestion,uptakeofnutrients,and antimicrobialactivity.

Modulateintestinalimmuneresponses.

Regulateintestinalepithelialcells.YanandPolk,CurrOpinGastroenterol20:565-571,2004扁平細(xì)胞樹突狀細(xì)胞基底膜上皮緊密連接粘液促進(jìn)腸道發(fā)育提高多糖的消化和營養(yǎng)物質(zhì)的吸收，提高抗菌活性，從而有益于宿主健康調(diào)節(jié)腸道免疫反應(yīng)調(diào)節(jié)腸道上皮細(xì)胞ThemechanismsofprobioticactionBlockingpathogenicbacterialeffects.

Modulatingmucosalimmuneresponses.

Promotingintestinalepithelialintegrity,survivalanddefenseresponses.

Vanderpooletal.,InflamBowelDis,2008Probiotics:Livemicroorganismswhichwhenconsumedinadequateamounts aspartoffood,conferahealthbenefittothehost.阻止致病菌的入侵調(diào)節(jié)粘膜免疫反應(yīng)促進(jìn)腸道上皮的完整性、存活以及防御反應(yīng)LactobacillusrhamnosusGG(LGG)

IsolatedfromahealthyhumanOneofthebest-studiedprobioticbacteriainclinicaltrialtopreventand/ortreat: Diarrhea,Clostridiumdifficile,RotavirusandEnterococciinfections Ulcerativecolitis,AtopicdermatitisLGG-derivedsolublefactors: Intestinalepithelialcells: Survivalandproliferation Barrierfunction Synthesisofcytoprotectiveproteins Macrophages: DecreaseLPS-inducedTNFproduction

IncreaseG-CSF

LGG巨噬細(xì)胞腸上皮細(xì)胞鼠李糖乳桿菌GG株在預(yù)防和治療輪狀病毒腹瀉、梭狀芽孢桿菌、腸球菌感染的潰瘍性結(jié)腸炎，過敏性皮炎的臨床試驗中，它是研究的最多的益生菌之一。細(xì)胞保護(hù)蛋白的合成細(xì)菌脂多糖粒細(xì)胞集落刺激因子腫瘤壞死因子LGGandfactorsrecoveredfromLGGbrothculturesupernatantregulateintestinalepithelialcellsignalingpathwaystoblockapoptosis

LGG和從LGG肉湯培養(yǎng)基的上清液分離的因子通過調(diào)節(jié)腸上皮細(xì)胞的信號轉(zhuǎn)導(dǎo)

途徑來阻止細(xì)胞凋亡YanandPolk,J.Biol.Chem.,52,50959,2002.

(pro-apoptotic)(Anti-apoptotic)ApoptosisTNFIFNIL-1ColonepithelialcellCytokineReceptorLGGLGGsolubleproteinsAktp38Cytokinereceptor細(xì)胞因子受體Colonepithelialcell結(jié)腸上皮細(xì)胞Apoptosis細(xì)胞凋亡Pro-apoptotic促凋亡Anti-apoptotic抗凋亡Apoptosis細(xì)胞凋亡Whyisitimportanttodevelopbacteria-derivedsolubleproteinsforclinicalapplications?為什么開發(fā)細(xì)菌衍生的可溶性蛋白的臨床應(yīng)用是很重要的？Concernsforuseofprobioticbacteriaintherapies:益生菌用于治療的關(guān)注點1、Bioavailability–survival,locationandfunctionofprobioticbacteriainthegastrointestinaltractrelativelyunknown

生物利用率——益生菌在胃腸道中的存活率，位置和功能都是未知的2、Biosafety(youngandimmuno-compromisedpatients)

安全性（小孩和免疫系統(tǒng)有缺陷的人群）Approachtoaddresstheseconcerns:-Developingprobioticbacteria-derivedproteinsfortargeteddelivery

開發(fā)能夠定向投放的益生菌衍生蛋白 TodefinemechanismsthroughwhichLGG-secretedsolubleproteinsregulateintestinalepithelialcellfunction

確定LGG分泌的哪種可溶性蛋白調(diào)節(jié)腸道上皮細(xì)胞功能的機制Aim1Purificationofp40andp75fromLGGbrothculturesupernatant(LGG-s)p40p75Yanetal.,Gastroenterology,132,562,2007p40andp75activationofAkt顆粒過濾器LGG分泌蛋白陽離子交換色譜NaCl洗脫p40andp75preventTNF-inducedapoptosisincolonepithelialcells.在結(jié)腸上皮細(xì)胞p40和p75阻止TNF-α誘導(dǎo)細(xì)胞凋亡Yanetal.,Gastroenterology,132,562,2007

*p<0.01comparedtoTNFtreatment1、TUNEL法：[terminaldexynucleotidyltransferase(TdT)-mediateddUTPnickendlabeling](末端脫氧核苷酸轉(zhuǎn)移酶介導(dǎo)的dUTP缺口末端標(biāo)記測定法)也稱DNA斷裂的原位末端標(biāo)記法，這一方法能對，DNA分子斷裂缺口中的3’-OH進(jìn)行原位標(biāo)記，借助一種可觀測的標(biāo)記物，如熒光素，能對凋亡細(xì)胞的核DNA中產(chǎn)生的3’-OH末端進(jìn)行原位標(biāo)記，用熒光顯微鏡即可進(jìn)行觀察。2、DAPI：DAPI即4',6-二脒基-2-苯基吲哚(4',6-diamidino-2-phenylindole)，是一種能夠與DNA強力結(jié)合的熒光染料，常用與熒光顯微鏡觀測1。因為DAPI可以透過完整的細(xì)胞膜，它可以用于活細(xì)胞和固定細(xì)胞的染色。EGFreceptor(EGFR)signalingpathwayregulatesAktactivationandcellularresponses.

表皮生長因子受體（EGFR）信號通路的調(diào)節(jié)Akt的活化和細(xì)胞的反應(yīng)

EGFR:Type1receptortyrosinekinaseExpressedbasolaterallyintheintestinalepitheliumcolonepithelialcell結(jié)腸上皮細(xì)胞proliferation增殖migration遷移differentiation區(qū)別survival生存RTKsGPCRG蛋白歐聯(lián)受體Cytokine細(xì)胞因子Receptor接收器srcMMPs基質(zhì)金屬蛋白酶Ligands配體trans-membraneligands跨膜配體EGFR表皮生長因子受體絲裂原活化蛋白激酶胞內(nèi)磷脂酰肌醇激酶，與v．src和v．ras等癌基因的產(chǎn)物相關(guān)，且PI3K本身具有絲氨酸/蘇氨酸(Ser/Thr)激酶的活性，也具有磷脂酰肌醇激酶的活性EGFR為I型跨膜酪氨酸激酶生長因子受體．定位于細(xì)胞膜．與HER2／ErbB．2／Neu／pl85、HER3／ErbB一3、HER4／ErbB-4等同歸入HER／Eerb家族。由胞外區(qū)、跨膜區(qū)及胞內(nèi)區(qū)三部分組成。其配體包括表皮生長因子(epidermalgrowthfactor。EGF)、轉(zhuǎn)化生長因子Of．(transforminggrowthfactor-or，TGF—d)、8一細(xì)胞素(betacellulin，BTC)、雙調(diào)蛋白(amphiregulin)、表皮素(epiregulin)、肝素結(jié)合的表皮生長因子(heparin．bindingEGF，HB．EGF)等，以EGF和TGF．d最為重要。EGFR與其配體胞外區(qū)結(jié)合后相互作用形成同源二聚體或異源二聚體。與同源二聚體相比，異源二聚體在介導(dǎo)細(xì)胞增殖、分化、遷移等信號傳遞中起著更為重要的作用。二聚體的形成導(dǎo)致胞內(nèi)酪氨酸激酶區(qū)的激活，進(jìn)而通過轉(zhuǎn)磷酸化和磷酸化作用，促使受體酪氨酸殘基磷酸化．啟動ras．MAPK、P13K、PLC'y／PKC、STAT等一系列級聯(lián)反應(yīng)．將信號傳到細(xì)胞核內(nèi)．最終引起一系列相關(guān)基因活化．促進(jìn)細(xì)胞從G，期過渡到S期141．從而對核內(nèi)基因表達(dá)和細(xì)胞生長分化產(chǎn)生調(diào)節(jié)作用。p40activatesAktinanEGFRdependentmannerinintestinalepithelialcells.在腸上皮細(xì)胞p40依靠EGFR（表皮生長因子）受體激活A(yù)kt蛋白EGFRisrequiredforp40preventionofcytokine-inducedapoptosisinintestinalepithelialcells在腸上皮細(xì)胞中預(yù)防細(xì)胞因子誘導(dǎo)的凋亡，EGFR對P40來說是必需的p40orp75preventH2O2disruptionoftightjunctioninCaco2cells.p40或p75可以防止H2O2中斷Caco2細(xì)胞緊密連接

H2O2H2O2/p40H2O2/p40/AG1478H2O2/p75H2O2/p75/AG1478ZO-1OccludinAG1478:EGFreceptorkinaseinhibitorAG1478：表皮生長因子受體激酶抑制劑Sethetal.AmJPhysGastrointestLiverPhysiol2008p40requiresEGFRkinaseactivitytostimulateAktactivationinmousecolonexplants.在小鼠結(jié)腸外植體中，p40需要EGFR激酶活性刺激Akt的活性wt(C57BL/6)EGFRwa5p40(10ng/ml)ColonexplantDMEM0.5%FBSColonicmucosallysatesEGFRkinaseinhibitorAG1478(10nM)結(jié)腸黏膜裂解p40inhibitionofTNF-inducedapoptosisrequiresEGFRactivityincolonicorganculture.在結(jié)腸器官培養(yǎng)物中，p40抑制TNF-α誘導(dǎo)的細(xì)胞凋亡需要EGFR的活性EGFRmediatesp40preventionofTNF-inducedredistributionofoccludininintestinalepithelialcells.在腸上皮細(xì)胞中，EGFR介導(dǎo)p40預(yù)防TNF-α誘導(dǎo)的密封蛋白的再分配Red:Occludin,

Blue:DAPIControlTNFTNF+p40wtEGFRwa5紅色代表Occludin密封蛋白藍(lán)色：DAPIp40preventscytokine-inducedapoptosisanddisruptionofintegritythroughactivationofEGFreceptor(EGFR)andAktinintestinalepithelialcells.在腸上皮細(xì)胞p40通過激活表皮生長因子受體（EGFR）和Akt，阻止細(xì)胞因子誘導(dǎo)的細(xì)胞凋亡和破壞其完整性。Yanetal.Gastroenterology32:562-75,2007,JClinInvest121:2242-53,2011.Colonepithelialcell

AktActivationEGFRTNFIFNIL-1CytokineReceptorApoptosisDisruptionofbarrierp40?Aim2Toinvestigatewhetherp40stimulatesEGFreceptorligandreleaseasamechanismfortransactivationofEGFreceptorbyp40intheintestinalepithelialcells.p40isanovelprotein

p40是一種新的蛋白

Full-lengthp40proteinsequence(412aa)

MKFNKAMITLVAAVTLAGSVSALTPVFADTSASIASNKSETNDLLKQIEAANTEVINLNKQIDAKNGEISDATAKISATDAKIASLSGEITAAQKNVAARKNNLKDQLISLQKKAGSSVSGNVYIDFVLNSQSLSDLIARTMTVGKLSQASKDALDAVTVAKDKLAALKSEQETARQTLVSTKASLETQKSQLETLQKTASDKQDALNKEIADHKDELVALQSQFAQEQSEAAKATQAALKTAAASTASSSTSSTSNKSANSSVLSTGTSSTNTSSNSGASSTVISSNTASGSGSHADYSGSGNTYPWGQCTWYVKSVASWAGNGWGNGAEWGASAAAAGFTVNHTPAAGSIIVFAAGQSVGGQWTADGSYGHVAYVQSVSGDSVTITQGGMGFSSPTGPNTQTISGASSYVGreen:N-terminalsequencedetectedbyEdmandegradation.Orange:internalsequencedetectedbyMALDI-TOF/MS/MSandLC/MS/MSanalysis.Purple:Leadersequenceforproteinsecretion.綠：N-末端序列由Edman降解法檢測。橙：由MALDI-TOF/MS/MS和LC/MS/MS分析檢測到的內(nèi)部序列。紫色：前導(dǎo)序列的蛋白質(zhì)分泌。Yanetal.,Gastroenterology,132,562,2007Predictionofp40tertiarystructureforstructure-functionanalysisp40的三級結(jié)構(gòu)的結(jié)構(gòu)與功能分析預(yù)測p40aminoacidsequencehomologyanalysisHigh-confidencetemplatesforp40domainsUncharacterizeddomain-sheetcoiled-coilNH2175150225300375412COOHp40的氨基酸序列的同源性分析高信任度的p40域模板p40stimulatesHB-EGFrelease,butnotTGForamphiregulin,inyoungadultmousecolon(YAMC)epithelialcells.在年輕成年小鼠結(jié)腸（YAMC的）上皮細(xì)胞，p40刺激HB-EGF的釋放，而不是TGF或雙調(diào)蛋白。(10ng/ml)YAMCp40Supernatant:ELISALysates:WesternblotHB-EGF上清p40stimulatesHB-EGFrelease,butnotTGForamphiregulin,inmice在小鼠實驗中，P40刺激HB-EGF的釋放，而不是TGF或雙調(diào)蛋白Gavagep40-pectin/zeinbeadsBloodELISAKnock-downHB-EGFsuppressesactivationofEGFRandinhibitionofapoptosisbyp40inYAMCcells.在YAMC細(xì)胞中去除HB-EGF通過p40抑制EGFR的活性并抑制細(xì)胞凋亡Metalloproteinaseinhibitorsblockp40-stimulatedEGFRactivationinYAMCcells.在YAMC細(xì)胞中，金屬蛋白酶抑制劑阻止p40刺激表皮生長因子的活性ADAM:ADisintegrinAndMetalloproteinasep40:10ng/ml,1hrEGF:10ng/ml,5minMetalloproteinaseinhibitors:GM6001(10nM)andTAPI-1(10nM)Anti-EGFRantibody:C225,1:1000dilutionInactive

MembraneboundligandactivePropeptideMetalloproteaseDisintegrinCystein-richTransmembranecytoplasmic

ADisintegrinAndMetalloproteinase去整合素和金屬蛋白酶inactive無活性propeptide肽Metalloprotease金屬蛋白酶Disintegrin整合素cystein-rich富含半胱氨酸Transmembrane跨膜Cytoplasmic細(xì)胞質(zhì)membraneboundligand膜結(jié)合配體p40stimulatesTACE(ADAM17)activityincolonepithelialcellsinvitroandinvivo.在體內(nèi)或體外的結(jié)腸上皮細(xì)胞中，p40刺激TACE（肝動脈栓塞？）（ADAM17）的活性YAMCp40Lysates:TACEactivityassayGavagep40-pectin/zeinbeadsColonicepithelialcellsTACEactivityassayHouraftergavageTACEmediatesp40regulatedanti-apoptoticresponseincolonepithelialcells.在結(jié)腸上皮細(xì)胞上，TACE介導(dǎo)P40的抗凋亡性反應(yīng)p40transactivationofEGFRdependsonTACE.TACEmediatesinhibitionofapoptosisbyp40.P40的表皮生長因子受體的轉(zhuǎn)錄依賴于TACE通過p40，TACE介導(dǎo)抑制細(xì)胞凋亡Summaryp40-stimulatedTACEactivity,leadingtoHB-EGFrelease,servesasamechanismunderlyingEGFreceptortransactivationinintestinalepithelialcells在腸上皮細(xì)