1、Fang Yan, M.D., Ph.D. Department of PediatricsDivision of Gastroenterology, Hepatology and NutritionVanderbilt University School of MedicineNashville, TN,Support: National Institute of Diabetes and Digestive Diseases and Kidney Diseases, Crohns and Colitis Foundation of America, Vanderbilt Digestive
2、 Disease Research Center, the Nestle Nutrition Grant, and Atticus-Brown Family Trust,Probiotic-derived soluble factors regulate host defense mechanisms: impact on digestive health,糖尿病、消化系统疾病和肾病国家研究所、美国克洛病和结肠炎基金会、范德比特消化系统疾病研究中心、雀巢营养公司的授权、布朗家族信托,益生菌衍生的可溶性因子对于宿主防御的调节机制：对消化系统的健康影响,范德比特大学医学院肠道学、肝胆学和营养学部门
3、儿科田纳西州纳什维尔,Research projects:1. Prevention and treatment of intestinal inflammation - Probiotic (Lactobacillus GG)-derived soluble proteins- Berberine (an alkaloid isolated from plants) 2. The roles of EGF and TNF signaling pathways in regulation of injury and repair mechanisms in intestinal inflamm
4、ation and carcinogenesis3. The mechanisms of H.pylori-induced gastric inflammation and cancer development,预防和治疗肠道炎症-LGG的分泌蛋白-黄连素（一种从植物中分离的生物碱）-可治疗腹泻,EGF和TNF的信号通路在肠道炎症及癌变中对损伤和修复机制的调节作用。,幽门螺杆菌诱导胃炎症和癌变的机制,Intestinal epithelial cell homeostasis and functions肠上皮细胞的动态平衡和功能,Digestion and absorption Water a
5、nd electrolyte transport Protective defense mechanisms Barrier function (tight junctions) Anti-bacterial substancesInnate and adaptive immunity,肠细胞,保护防御机制,屏障功能,腺管,小肠绒毛,杯状细胞,潘氏细胞,内分泌细胞,干细胞,先天免疫后天免疫,抗菌物质,The gastrointestinal microbiota,杆菌属真菌属双歧杆菌属瘤胃球菌属芽孢杆菌属梭菌属乳杆菌属肠球菌属肠杆菌属,胃肠道细菌,幽门螺杆菌,Commensal bacteri
6、a in the gastrointestinal tract胃肠道中的共生细菌,Beneficial effects: Promote intestinal development. Benefit host health by enhancing polysaccharide digestion, uptake of nutrients, and antimicrobial activity. Modulate intestinal immune responses. Regulate intestinal epithelial cells.,Yan and Polk, Curr Opin
7、 Gastroenterol 20: 565-571, 2004,扁平细胞,树突状细胞,基底膜,上皮,紧密连接,粘液,促进肠道发育,提高多糖的消化和营养物质的吸收，提高抗菌活性，从而有益于宿主健康,调节肠道免疫反应,调节肠道上皮细胞,The mechanisms of probiotic action,Blocking pathogenic bacterial effects. Modulating mucosal immune responses. Promoting intestinal epithelial integrity, survival and defense response
8、s.,Vanderpool et al., Inflam Bowel Dis, 2008,Probiotics: Live microorganisms which when consumed in adequate amounts as part of food, confer a health benefit to the host.,阻止致病菌的入侵,调节粘膜免疫反应,促进肠道上皮的完整性、存活以及防御反应,Lactobacillus rhamnosus GG (LGG),Isolated from a healthy human One of the best-studied prob
9、iotic bacteria in clinical trial to prevent and/or treat: Diarrhea, Clostridium difficile, Rotavirus and Enterococci infections Ulcerative colitis, Atopic dermatitisLGG-derived soluble factors:Intestinal epithelial cells: Survival and proliferation Barrier function Synthesis of cytoprotective protei
10、nsMacrophages: Decrease LPS-induced TNF production Increase G-CSF,LGG,巨噬细胞,肠上皮细胞,鼠李糖乳杆菌GG株,在预防和治疗轮状病毒腹泻、梭状芽孢杆菌、肠球菌感染的溃疡性结肠炎，过敏性皮炎的临床试验中，它是研究的最多的益生菌之一。,细胞保护蛋白的合成,细菌脂多糖,粒细胞集落刺激因子,肿瘤坏死因子,LGG and factors recovered from LGG broth culture supernatant regulate intestinal epithelial cell signaling pathways
11、to block apoptosis LGG和从LGG肉汤培养基的上清液分离的因子通过调节肠上皮细胞的信号转导途径来阻止细胞凋亡,Yan and Polk, J. Biol. Chem., 52, 50959, 2002.,Cytokine receptor 细胞因子受体Colon epithelial cell 结肠上皮细胞Apoptosis 细胞凋亡Pro-apoptotic 促凋亡Anti-apoptotic 抗凋亡Apoptosis 细胞凋亡,Why is it important to develop bacteria-derived soluble proteins for cli
12、nical applications?为什么开发细菌衍生的可溶性蛋白的临床应用是很重要的？,Concerns for use of probiotic bacteria in therapies:益生菌用于治疗的关注点1、Bioavailability survival, location and function of probiotic bacteria in the gastrointestinal tract relatively unknown 生物利用率益生菌在胃肠道中的存活率，位置和功能都是未知的2、Biosafety (young and immuno-compromised
13、patients) 安全性（小孩和免疫系统有缺陷的人群）Approach to address these concerns:- Developing probiotic bacteria-derived proteins for targeted delivery 开发能够定向投放的益生菌衍生蛋白,To define mechanisms through which LGG-secreted soluble proteins regulate intestinal epithelial cell function 确定LGG分泌的哪种可溶性蛋白调节肠道上皮细胞功能的机制,Aim 1,Puri
14、fication of p40 and p75 from LGG broth culture supernatant (LGG-s),Yan et al., Gastroenterology, 132, 562, 2007,p40 and p75 activation of Akt,颗粒过滤器,LGG分泌蛋白,阳离子交换色谱,NaCl洗脱,p40 and p75 prevent TNF-induced apoptosisin colon epithelial cells.在结肠上皮细胞p40和p75阻止TNF-诱导细胞凋亡,Yan et al., Gastroenterology, 132,
15、562, 2007,* p 0.01 compared to TNF treatment,1、TUNEL 法：terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick end labeling(末端脱氧核苷酸转移酶介导的dUTP 缺口末端标记测定法 ) 也称DNA 断裂的原位末端标记法，这一方法能对，DNA分子断裂缺口中的3-OH进行原位标记，借助一种可观测的标记物，如荧光素，能对凋亡细胞的核DNA中产生的3-OH 末端进行原位标记，用荧光显微镜即可进行观察。2、DAPI：DAPI即4,6-二脒基-2-苯基吲哚(4,6-diami
16、dino-2-phenylindole)，是一种能够与DNA强力结合的荧光染料，常用与荧光显微镜观测1。因为DAPI可以透过完整的细胞膜，它可以用于活细胞和固定细胞的染色。,EGF receptor (EGFR) signaling pathway regulates Akt activation and cellular responses.表皮生长因子受体（EGFR）信号通路的调节Akt的活化和细胞的反应,EGFR: Type 1 receptor tyrosine kinase Expressed basolaterally in the intestinal epithelium,co
17、lon epithelial cell结肠上皮细胞proliferation增殖migration迁移differentiation区别survival生存RTKsGPCR G蛋白欧联受体Cytokine细胞因子Receptor接收器srcMMPs基质金属蛋白酶Ligands配体trans-membrane ligands 跨膜配体EGFR表皮生长因子受体,丝裂原活化蛋白激酶,胞内磷脂酰肌醇激酶，与vsrc和vras等癌基因的产物相关，且PI3K本身具有丝氨酸/苏氨酸(Ser/Thr)激酶的活性，也具有磷脂酰肌醇激酶的活性,EGFR为I型跨膜酪氨酸激酶生长因子受体定位于细胞膜 与H ER2Er
18、bB2Neup l 85、HER3ErbB一3、HER4ErbB-4等同归入HEREerb家族。由胞外区、跨膜区及胞内区三部分组成。其配体包括表皮生长因子(epidermal growth factor。EGF)、转化生长因子Of(transforming growth factor-or，TGFd)、8一细胞素(betacellulin，BTC)、双调蛋白(amphiregulin)、表皮素(epiregulin)、肝素结合的表皮生长因子(heparinbinding EGF，HBEGF)等，以EGF和TGFd最为重要。EGFR与其配体胞外区结合后相互作用形成同源二聚体或异源二聚体。与同源二
19、聚体相比，异源二聚体在介导细胞增殖、分化、迁移等信号传递中起着更为重要的作用。二聚体的形成导致胞内酪氨酸激酶区的激活，进而通过转磷酸化和磷酸化作用，促使受体酪氨酸残基磷酸化启动rasMAPK、P13K、PLCyPKC、STAT等一系列级联反应将信号传到细胞核内最终引起一系列相关基因活化促进细胞从G，期过渡到S期141从而对核内基因表达和细胞生长分化产生调节作用。,p40 activates Akt in an EGFR dependent manner in intestinal epithelial cells.在肠上皮细胞p40依靠EGFR（表皮生长因子）受体激活Akt蛋白,EGFR is
20、 required for p40 prevention of cytokine-induced apoptosis in intestinal epithelial cells在肠上皮细胞中预防细胞因子诱导的凋亡，EGFR对P40来说是必需的,p40 or p75 prevent H2O2 disruption of tight junction in Caco2 cells.p40或p75可以防止H2O2中断Caco2细胞紧密连接,AG1478: EGF receptor kinase inhibitorAG1478：表皮生长因子受体激酶抑制剂,Seth et al. Am J Phys
21、Gastrointest Liver Physiol 2008,p40 requires EGFR kinase activity to stimulate Akt activation in mouse colon explants.在小鼠结肠外植体中，p40需要EGFR激酶活性刺激Akt的活性,结肠黏膜裂解,p40 inhibition of TNF-induced apoptosis requires EGFR activity in colonic organ culture.在结肠器官培养物中，p40抑制TNF-诱导的细胞凋亡需要EGFR的活性,EGFR mediates p40 p
22、revention of TNF-induced redistribution of occludin in intestinal epithelial cells.在肠上皮细胞中，EGFR介导p40预防TNF-诱导的密封蛋白的再分配,红色代表Occludin 密封蛋白 蓝色：DAPI,p40 prevents cytokine-induced apoptosis and disruption of integrity through activation of EGF receptor (EGFR) and Akt in intestinal epithelial cells.在肠上皮细胞p
23、40通过激活表皮生长因子受体（EGFR）和Akt，阻止细胞因子诱导的细胞凋亡和破坏其完整性。,Yan et al. Gastroenterology 32:562-75, 2007, J Clin Invest 121:2242-53, 2011.,Colon epithelial cell,Akt,Activation,EGFR,p40 is a novel proteinp40是一种新的蛋白,Full-length p40 protein sequence (412 aa) MKFNKAMITLVAAVTLAGSVSALTPVFADTSASIASNKSETNDLLKQIEAANTEVINL
24、NKQIDAKNGEISDATAKISATDAKIASLSGEITAAQKNVAARKNNLKDQLISLQKKAGSSVSGNVYIDFVLNSQSLSDLIARTMTVGKLSQASKDALDAVTVAKDKLAALKSEQETARQTLVSTKASLETQKSQLETLQKTASDKQDALNKEIADHKDELVALQSQFAQEQSEAAKATQAALKTAAASTASSSTSSTSNKSANSSVLSTGTSSTNTSSNSGASSTVISSNTASGSGSHADYSGSGNTYPWGQCTWYVKSVASWAGNGWGNGAEWGASAAAAGFTVNHTPAAGSIIVFAAG
25、QSVGGQWTADGSYGHVAYVQSVSGDSVTITQGGMGFSSPTGPNTQTISGASSYV,Green: N-terminal sequence detected by Edman degradation.Orange: internal sequence detected by MALDI-TOF/MS/MS and LC/MS/MS analysis.Purple: Leader sequence for protein secretion.绿：N-末端序列由Edman降解法检测。橙：由MALDI-TOF/MS/MS和LC/ MS / MS分析检测到的内部序列。紫色：前导
26、序列的蛋白质分泌。,Yan et al., Gastroenterology, 132, 562, 2007,Prediction of p40 tertiary structure for structure-function analysisp40的三级结构的结构与功能分析预测,p40 amino acid sequence homology analysis,High-confidence templates for p40 domains,Uncharacterized domain,-sheet,p40的氨基酸序列的同源性分析,高信任度的p40域模板,p40 stimulates H
27、B-EGF release, but not TGF or amphiregulin, in young adult mouse colon (YAMC) epithelial cells.在年轻成年小鼠结肠（YAMC的）上皮细胞，p40刺激HB-EGF的释放，而不是TGF或双调蛋白。,(10 ng/ml),上清,p40 stimulates HB-EGF release, but not TGF or amphiregulin, in mice在小鼠实验中，P40刺激HB-EGF的释放，而不是TGF或双调蛋白,Knock-down HB-EGF suppresses activation o
28、f EGFR and inhibition of apoptosis by p40 in YAMC cells.在YAMC细胞中去除HB-EGF通过p40抑制EGFR的活性并抑制细胞凋亡,Metalloproteinase inhibitors block p40-stimulated EGFR activation in YAMC cells.在YAMC细胞中，金属蛋白酶抑制剂阻止 p40 刺激表皮生长因子的活性,ADAM: A Disintegrin And Metalloproteinase,p40: 10 ng/ml, 1 hrEGF: 10 ng/ml, 5 minMetallopr
29、oteinase inhibitors: GM6001 (10 nM) and TAPI-1 (10 nM) Anti-EGFR antibody: C225, 1:1000 dilution,A Disintegrin And Metalloproteinase 去整合素和金属蛋白酶inactive 无活性propeptide肽Metalloprotease 金属蛋白酶Disintegrin 整合素cystein-rich 富含半胱氨酸Transmembrane 跨膜Cytoplasmic 细胞质membrane bound ligand 膜结合配体,p40 stimulates TACE
30、(ADAM17) activity in colon epithelial cells in vitro and in vivo.在体内或体外的结肠上皮细胞中，p40刺激TACE（肝动脉栓塞？）（ADAM17）的活性,Gavage p40-pectin/zein beads,Colonic epithelial cells,TACE activity assay,Hour after gavage,TACE mediates p40 regulated anti-apoptotic response in colon epithelial cells.在结肠上皮细胞上，TACE介导P40的抗凋
31、亡性反应,p40 transactivation of EGFR depends on TACE.,TACE mediates inhibition of apoptosis by p40.,P40的表皮生长因子受体的转录依赖于TACE,通过p40，TACE介导抑制细胞凋亡,Summary,p40-stimulated TACE activity, leading to HB-EGF release, serves as a mechanism underlying EGF receptor transactivation in intestinal epithelial cells在肠上皮细
32、胞，P40-刺激TACE的活性，导致HB-EGF释放，从而使其在表皮生长因子受体下转录,Aim 3,To test the hypothesis that p40 prevents experimental colitis in mice in an EGFR-dependent manner.为了检验这个假设，p40依赖表皮生长因子受体来抑制小鼠的实验性结肠炎,Preparation of pectin/zein beads for specific delivery of p40 to the colon in vivo. 制备果胶/玉米醇溶蛋白的药丸（包埋有p40），能够使其在体内到达结
33、肠位置。,Gavaged with pectin/zein beads (2 beads/mouse) containing FITC-His-p40 or His-p40- Sacrificed after 4 hours.,Yan et al., JCI, 121, 2242, 2011,-灌胃果胶/玉米醇溶蛋白的药丸（每只小鼠两颗），药丸里面包含有FITC-His-p40（标记的）或His-p40（未标记的）-药丸在4h后完全消化,上表皮钙粘着蛋白,p40 activates EGFR and Akt in colon epithelial cells in vivo.在体内的结肠上皮细
34、胞中，p40激活EGFR和Akt,Yan et al., JCI, 121, 2242, 2011,磷酸化表皮生长因子受体,p40 prevents DSS-induced colitis in wt, but not EGFRwa2 mice.在wt小鼠模型中p40防止DSS诱导结肠炎，但在EGFRwa2小鼠模型中不起作用,Yan et al., JCI, 121, 2242, 2011,损伤或炎症得分,牺牲/处理,P40 treats DSS-induced colitis in wt, but not EGFRwa2 mice.在wt小鼠模型中p40可以治疗DSS诱导的结肠炎，但在EGF
35、Rwa2小鼠模型中不起作用,Yan et al., JCI, 121, 2242, 2011,p40 blocks DSS-induced colon epithelial cell apoptosis in an EGFR-dependent manner.p40需要依赖EGFR来抑制DSS诱导结肠上皮细胞凋亡,Yan et al., JCI, 121, 2242, 2011,半胱氨酸天冬氨酸蛋白酶-3：被激活后可以通过对caspase靶蛋白的水解，导致程序性细胞死亡,Bcl-2基因（即B细胞淋巴瘤/白血病-2基因）是一种原癌基因，它具有抑制凋亡的作用,p40 prevents DSS-in
36、duced disruption of intestinal epithelial tight junctions in an EGFR-dependent manner. p40需要依赖EGFR来抑制DSS诱导肠上皮细胞紧密连接的中断,Yan et al., JCI, 121, 2242, 2011,体内渗透检测,血清FITC-葡聚糖,紧密连接蛋白ZO-1,Generation of transgenic mice with EGF receptor specifically deleted in the intestinal epithelium.新一代的转基因小鼠的肠上皮细胞中专门去除E
37、GFR,Yan et al., JCI, 121, 2242, 2011,上皮,基质,EGF receptor expression in the colon epitheliumis required for p40 treating DSS-induced colitis in mice.p40治疗DSS诱导的小鼠结肠炎需要EGFR在结肠上皮细胞的表达,Yan et al., JCI, 121, 2242, 2011,外伤/炎症评分,血清中FITC标记的聚葡萄糖,凋亡细胞所占百分比,p40 ameliorating oxazolone-induced colitis requires EG
38、F receptor expression in the colon epithelium.p40改善恶唑酮诱导的小鼠结肠炎需要EGFR在结肠上皮细胞的表达,乙醇,p40 activation of EGF receptor in intestinal epithelial cells mediates amelioration of intestinal inflammation in mice.在肠上皮细胞中p40激活EGFR的活性，间接地改善了小鼠肠道炎症,Yan et al., J Clin Invest 121:2242-53, 2011,破坏屏障,炎症,Aim 4To detect
39、 immune responses regulated by p40 during colitis in mice.检测小鼠结肠炎期间由p40调控的免疫反应,p40 treatment decrease macrophage and neutrophil infiltration in DSS-induced colitis in mice.在具有DSS诱导的结肠炎的小鼠中，p40处理后可以减少巨噬细胞和嗜中性白血球的渗透活动,嗜中性白血球,巨噬细胞,p40 treatment decreases TNF, IFN-, IL-6, KC, and IL-17 mRNA levels in DS
40、S treated mouse colon mucosa.在DSS处理的结肠粘膜的小鼠中，p40处理后可以减少TNF, IFN-, IL-6, KC, 和IL-17 mRNA,巨噬细胞产生TNF, IL-6和KC，TH1细胞产生IFN-,p40 treatment does not affect IL-10, IL-1 mRNA levels in DSS treated mouse colon mucosa.在DSS处理的结肠粘膜的小鼠中，p40处理后不影响IL-10，IL-1mRNA水平,IL-10、IL-13主要由th2细胞产生,p40 does not affect IL-13 pro
41、duction in oxazolone-induced colitis in mice.在具有恶唑酮诱导的结肠炎的小鼠中，p40处理后不影响IL-13的量,Colon mucosal IL-13 mRNA levelReal time PCR,Colon mucosal IL-13 protein levelFlow cytometry,实时定量PCR,流式细胞仪,结肠黏膜IL-13蛋白水平,结肠黏膜IL-13 mRNA水平,IL-13由th2细胞产生,SummaryBased on the cytokine profiles that are regulated by p40, it is
42、 possible that p40 plays a role in regulation of innate immunity and a Th1 immune response in colitis, such as regulation of TNF, IL-6 and KC production by macrophages and IFN- production by Th1 cells. 根据上述资料，p40可能在结肠炎中发挥先天免疫和Th1型的免疫应答的调节作用，如调节巨噬细胞产生TNF, IL-6和KC，TH1细胞产生IFN-。,Our findings support dev
43、elopment of probiotic-derived proteins as novel reagents for more convenient and direct therapeutic intervention in preventing and/or treating ulcerative intestinal inflammatory disorders. 我们的研究结果表明益生菌的衍生蛋白质可以可以作为一种新型的试剂更方便、更直接的治疗和预防溃疡性肠道炎症疾病。,Conclusion,Lactobacillus GG-fermented milk prevents DSS-
44、induced colitis and regulates intestinal epithelial homeostasis through activation of epidermal growth factor receptorLGG发酵乳可以通过调节EGFR的活性防止DSS诱导的结肠炎并能调节肠上皮细胞的动态平衡,p40 and p75 are present in probiotics-fermented milk products.在益生菌发酵乳制品中发现存在有p40和p75,LGG-fermented milk activates EGF receptor and Akt an
45、d prevents apoptosis in colon epithelial cells.LGG发酵乳能够激活EGFR和Akt的活性，并防止结肠上皮细胞的凋亡,HT-29,YAMC,PARP剪切被认为是细胞凋亡的一个重要指标，也通常被认为是Caspase 3激活的指标,EGFR抑制剂,放线菌酮阻碍蛋白质的合成,LGG-fermented milk preserves barrier function in colon epithelial cells.LGG发酵乳可以维护结肠上皮细胞的屏障功能,Red: ZO-1 staining,LGG-fermented milk prevents D
46、SS-induced colitis in mice.LGG发酵乳可以防止DSS诱发小鼠的结肠炎,SummaryThese results suggest that LGG-fermented milk may regulate intestinal epithelial homeostasis and potentially prevent intestinal inflammatory diseases through activation of EGFR by LGG-derived proteins,这些结果都表明了LGG发酵乳可以调节肠道上皮细胞的动态平衡，从而防止肠道炎疾病，这些都
47、是通过LGG源性蛋白激活EGFR的活性来实现的。,Acknowledgements,Vanderbilt UniversityKeith Wilson, M.D.Richard M. Peek, M.D.Kay Washington, M.D., Ph.D.Rupesh Chaturvedi, Ph.D.Timothy L. Cover, M.D.Jonathan Sheehan, Ph.D. David Friedman, Ph.D.Walter J. Chazin, Ph.D.Jens Meiler, Ph.D.Eric Skaar, Ph.D.Bob Whitehead, Ph.D.US
48、 Department of AgricultureLinShu Liu, Ph.D.Kevin Hicks, Ph.D.Arland Hotchkiss, Ph.D.,Vanderbilt UniversityHanwei Cao, B.S.Yan Shi, Ph.D.Liping Liu, M.D.Lihong Wang, Ph.D.Ning Lu, M.D., Ph.D.Hailong Cao, M.D., Ph.D.Childrens Hospital Los Angeles D. Brent Polk, M.D. All members in Polks labTakanashi Milk Products Co., Ltd. Fang He, Ph.D.,
