博士生课程5(1)-细胞因子

1、8-30KDCK-CKRs Cytockines are low-molecular-weight regulatory proteins or glycoproteins secreted by white blood cells and various cells (vascular endothelial cell, epidermic cell and fibroblast ) in body in response to a number of stimuli. CytokineMjmacrophageIL-8Just as hormones serve as messenger o

2、f the endocrine system, so cytokine sever as messengers of the immune system. Unlike endocrine hormones , though, which exert their effects over large distances, cytokines general act locally.Cytokines can exert different actionsIL-2Proliferation, survivalExample:ChemokinesLeukocyte recruitmentAcute

3、 phase proteins, IL-6TNFIL-33Properties of cytokines细胞因子调节IgG类别转换 多效性 拮抗性 重叠性协同性Biological effects of the cytokinesAnti-infection and antitumorImmune regulationApoptosisStimulating the proliferation and differentiation of hemopoietic stem cellsPromote the growth and differentiation of various cellsP

4、articipation and regulation of the inflammatory responsesCytokine abnormalities in the pathogenesis in certain diseases Cytokines during hematopoiesisinterleukin, IL （白细胞介素）colony stimulating factor,CSF（集落刺激因子）interferon,IFN（干扰素）tumor necrosis factor,TNF （肿瘤坏死因子）Chemokine, CK （趋化因子）growth factor,GF

5、（生长因子）CytokinesCytokines of the innate and adaptive immune systemCytokines of innate immune system TNF, IL-1 and IL-6: pleiotropic mediators of inflammation (activators of endothelium, immune cells, liver and brain)Chemokines: regulators of immune cell adhesion at infection sitesIL-12: inducer of IF

6、N-, increase of cytotoxic activityType I IFN: inducers of antiviral state InterleukinInterleukins are secreted by some leukocytes and act upon other leukocytes, IL-1IL-35 have been identified. Interleukin-1, IL-1The release of TNF-a by macrophages induces local protective effects, but TNF-a can have

7、 damaging effects when released systemically (septic shock).Tumor necrosis factor,TNFThe cytokines that induce directly apoptosis of tumor cells, including TNF, TNF (Lymphotoxin, LT） , FASL, CD70L, CD30L, CD40L, OX40L, TRAIL (TNF related apoptosis-inducing ligand) Chemokinechemokines, a group of low

8、-molecular-weight cytokines including interleukin 8, that affect chemotaxis and other aspects of leukocyte behavior. CX3CRCXCRCX3CCXCCK及CKR分类CCCCCRXCR1999年国际命名委员会根据CK的结构特征命名依据N端前两个半胱氨酸(Cys)的相对位置不同分为:IL-12 produced by macrophages and dendritic cells is a key cytokine to fight intracellular microbes (

9、bacteria and viruses)IL-12 induces IFN-, an activator of macrophages (killing of phagocytosed microbes) and Th1 cell differentiationIL-12 also increases cytotoxic activity of CD8 and NK cellsIL-12The cytokines that resist viral infection, and interfere viral replication, including IFN, IFN, IFN, IFN

10、, IFN ( type I ); IFN ( type II ) type I InterferonAntiviral effects of Type I Interferons (IFN-a/b)Integrative signaling of cytokines during innate immune responsesCytokines of adaptive immune systemIL-2: prototypic lymphocyte growth factorIL-4: inducer of Th2 cells, B cell differentitation factorI

11、FN-: inducer of Th1 cellsIL-2 (又称T细胞生长因子,TCGF) ： 细胞来源：主要由T细胞产生。 作用方式：以自分泌和旁分泌方式发挥效应。 主要生物学功能： （1）活化T细胞，促进细胞因子产生； （2）刺激NK细胞增殖，增强NK杀伤活性及产生 细胞因子；诱导LAK细胞产生 （3）促进B细胞增殖和分泌抗体； （4）激活巨噬细胞。IL-4 induces Isotype switching against extracellular pathogens, such as helminths and athropodesIL-4Pleiotropic activitie

12、s of IFN-IFN- is secreted by activated T cells after IL-12 stimulation during antimicrobial responses (intracellular bacteria and viruses)Th1 cells help B cell sotype switch to IgG for complement binding and Fc-binding on phagocytes strong macrophage activator Anti-inflammatory cytokinesIL-10IL-10:

13、counteracts IFN- by blocking IL-12 production by macrophages, and expression of costimulators and MHC IIIL-10-deficient mice develop inflammatory bowel disease Tr1TGF-TGF- is secreted by regulatory T cells (Th3) to decrease immune responsesThe TGF receptor is a dimer of ALK and TGF-RII which signal

14、through Smad transcription factorsAbsence of TGF or TGF-RII induces autoimmunity in miceAdditional key cytokines IL-5: activator of eosinophils, also inducing production of additional eosinophils from bone marrow progenitorsIL-13: similar to IL-4, but in addition stimulates mucus production and tiss

15、ue fibrosis in allergic diseases GM-CSF: promotes maturation of bone marrow cells to monocytes and dendritic cellsG-CSF: generated at site of infection but acting as endocrine hormone to mobilize neutrophils during inflammation (clinical use)Colony stimulating factor, CSFThe cytokines that stimulate

16、 hemopoietic cells to form cell colony, Participate hemopoietic function are called CSF, for example GM-CSF,G-CSF,M-CSF, Epo,Tpo是一类可介导不同类型细胞生长和分化的细胞因子。根据其功能和作用的靶细胞不同，分别命名为：表皮生长因子（epidermal growth factor, EGF)血管内皮生长因子（vascular endothelial growth factor, VEGF)成纤维细胞生长因子（fibroblast growth factor, FGF)神经

17、生长因子(nerve growth factor, NGF)血小板衍生的生长因子（platelet derived growth factor, PDGF) 肝细胞生长因子（hepatic growth factor, HGF)Growth factorVEGF Receptor Signaling Growth factors applied to angiogenesisVEGF promotes Tumor AngiogenesisTH1 and TH2 subsets secrete different cytokinesTH1- cell-mediated functions; in

18、flammationTH2- humoral immunityTH1 and TH2 subsets regulate each other CD4+ T cells isolation and differentiationSpleens were mechanically dissociated, followed by depletion of erythrocytes. Naive CD4+ T cells were isolated by negative selection. CD4+ T cells were cultured in medium supplemented wit

19、h anti-mouse CD3 and anti-mouse CD28. rmIL-2 plus anti-mouse IL-4 and anti-mouse IFN-antibodies (Th0 conditions) or anti-mouse IL-4 plus rmIL-2 and rmIL-12 (Th1 conditions) or rmIL-2, rmIL-4 plus anti-mouse IFN- antibody (Th2 conditions) were added. After 4 days, T cells were washed and reseeded on

20、anti-mouse CD3-coated plates in the absence or in the presence of anti-mouse CD28 for 24 hours, and cytokine secretion was determined by ELISA.The red line represents GATA3 staining, the black line corresponds to isotype control. 细胞因子受体分类主要包括免疫球蛋白受体超家族(1gRSF)、I型细胞因子受体家族、型细胞因子受体家族(干扰素受体家族)、型细胞因子受体家族(

21、肿瘤坏死因子受体家族，TNFRF)和七次跨膜受体家族(又称G蛋白偶联受体家族)Cytokine receptors belong to families of receptor proteins, each with a distinctive structureThe various members of the interleukin-1 (IL-1) family and their receptors 趋化因子受体（ GPCRs，G-protein coupled receptors ） 属于7次跨膜受体超家族，胞内环状结构与Gi/Go蛋白偶联胞外细胞膜 细胞质COOHNH2G 蛋白S

22、ignaling of cytokine receptorsType I/II cytokine receptors: Jak-STAT pathwayTNF receptors: TRAF (TNF-RII, CD40) or by death domains (TNF-RI, Fas)TIR-domain: Toll-like receptor signaling through IRAKReceptor-associated kinases: TGF- receptors, c-kitG-protein-coupled signalingClass I and Class II cyto

23、kine receptors signal by a rapid pathway using receptor-associated kinases to activate specific transcription factors.Jak-STAT signaling pathways, common to many cytokine receptors4 Jak proteins known: Jak1-3 and Tyk26 STATs known: STAT1-4, 5a, 5b and 6Receptor dimerization central for signalingComb

24、ination of Jak/STATs confers specificityJAK/STATs Used by Key CytokinesIL-5-EosinophilIL-1TNFGM-CSFM-CSFFGFPDGFVEGFIL-12IL-15IL-6LIFOSMChemokinesTGFIL-10IL-11IL-13sTNF-RIL-1raPRO-INFLAMMATORYANTI-INFLAMMATORYCytokine imbalance during inflammation CXCR4（T细胞）与CCR5（巨噬细胞） 是HIV感染靶细胞的共受体。细胞因子抑制剂 用于治疗炎症，自身

25、免疫病，移植排斥，休克细胞因子的临床应用进入临床实验的细胞因子抑制剂名称公司适应症临床阶段可溶性IL-1受体(干粉吸入剂)Immunex哮喘I/II期可溶性IL-1受体（注射剂）急性髓样白血病I期IL-1受体拮抗剂Amegen败血性休克(试验中止),类风湿关节炎完成III期可溶性IL-4受体Immunex哮喘I/II期抗IL-4人源化抗体SmithKline哮喘I/II期抗IL-5人源化抗体SmithKline哮喘II期可溶性TNF受体II-Fc融合蛋白Immunex类风湿关节炎，慢性心衰上市，I期可溶性TNF受体I-Fc融合蛋白Hoffmann-La Roch休克,类风湿关节炎,多发性硬化症

26、II/III期抗TNF单抗（infliximab）CentocorCrohns Disease上市人源化抗HER2 (EGFR2)单抗Genentech乳腺癌转移上市 DAB389-IL-2(IL-2免疫毒素)SeragenT细胞淋巴瘤, I型糖尿病,严重类风湿关节炎,牛皮癣,HIV感染上市I/II期DAB389-EGFSeragen肿瘤I/II期人源化抗VEGF单抗Genentech Inc.晚期肺癌，肠癌III期抗VEGFR2（KDR）嵌合抗体Albert LoBuglio转移性肠癌I期人源化抗IL-8抗体（ABX-IL8）AbgenixSevere psoriasisI/II期临床IL1

27、3-PE38QQR（IL-13免疫毒素）NeoPharm, Inc肾癌I期临床TNF反义寡核苷酸Isis类风湿关节炎，牛皮癣I期临床抗eotaxin抗体（CAT-213）Cambridge Antibody变态反应I期临床IL-1ra gene therapy on DBA/1 mice of collagen-induced arthritis cDNA librariesPCRTTTTTTTTAA+sequencingIL-1ra geneligateIL-1ra: IL-1 receptor antagonistAT clone system (Invitrogen Co.)AT clo

28、ne vectorSubcloneeukaryotic expression vectorspcI-neoEcoRIEcoRIEcoRICIPEcoRI+ligationpcI-IL-1raEcoRIEcoRIidentifiedPCRRestriction enzymepcDI-IL-1ratransfectionsynoviocyteCOS-7in vitroEXPRESSIONIL-1ra proteinELISAmRNApcDI-IL-1rain vivo (gene therapy)muscle injection.DBA/1 micetype II collageninducedD

29、BA/1 mice with arthritis gene gun细胞因子的临床应用 已批准上市的细胞因子基因工程药物名称适应症IFN白血病、Kaposi肉瘤、肝炎、癌症、AIDSIFN慢性肉芽肿、生殖器疣、过敏性皮炎、感染性疾病、类风湿关节炎G-CSF自身骨髓移植、化疗导致的粒细胞减少症、 AIDS、白血病、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、 AIDS、再生障碍性贫血、Epo慢性肾衰导致的贫血、癌症或癌症化疗导致的贫血、失血后贫血IL-2癌症、免疫缺陷、疫苗佐剂IFN多发性硬化症IL-11放化疗所致血小板减少症干细胞因子(SCF)与G-CSF联合应用于外周血干细

30、胞移植EGF外用药治疗烧伤、溃疡bFGF外用药治疗烧伤、外周神经炎正进行临床试验的细胞因子基因工程药物细胞因子名称适应症公司IL-10炎症,银屑病,Crohns 病,类风湿关节炎Immunex，Schering-PloughIL-12肿瘤，HIV感染，I型变态反应 GITpo血小板减少症GI，沈阳三生TNF变异体肿瘤国内多家企业Flt3/flk3 Ligand(FL)肿瘤ImmunexMyeloid Progenitor Inhibitory Factor-1 (MPIF-1)肿瘤大剂量化疗HGSTGF-慢性皮肤溃疡，多发性硬化症GenzymeKeratinocyte Growth Facto

31、r-2 (KGF-2)促进烧伤、慢性溃疡的伤口愈合；抗癌药物引起的粘膜损伤；炎症性肠道疾病HGSMIP-1 变异体肿瘤化疗的骨髓保护作用British BiotechOPG（osteoprotegerin）骨质疏松AmgenMulitkine（白细胞产生的细胞因子混合制剂）转移性肿瘤CEL-SCIB细胞刺激因子（BlyS）免疫缺陷HGS二、细胞因子的研究热点1、新细胞因子的基因克隆2、细胞因子受体的基因克隆3、细胞因子信号转导机制4、新一代细胞因子：高活性，多功能，低毒副作用，长半衰期，高稳定性5、细胞因子作为生物应答调节剂（BRM）的临床应用6、细胞因子表达调控7、细胞因子基因治疗1、新细胞

32、因子的基因克隆化从人类基因组发现新细胞因子后基因组时代的“捞鱼”2、可溶性细胞因子受体作为拮抗剂effectsNC-S-S-S-S-S-S-N-S-S-S-S-NC53DNAProtein（1）、细胞因子受体的克隆NC-S-S-S-S-S-S-53DNAPCR可溶性细胞因子受体expressN-S-S-S-S-Receptor blocker (as a receptor antagonist) N-S-S-S-S-N-S-S-S-S-N-S-S-S-S-NC-S-S-S-S-S-S-N-S-S-S-S-（2）、细胞因子受体的突变CN5353TAASite-directed mutagenes

33、isSoluble cytokine receptorCN细胞因子的检测1 生物学活性检测2 免疫学检测3 分子生物学检测1 生物学活性检测（1）趋化因子的检测显微镜下观察, 计数 : 细胞数/视野This scanning electron micrograph shows two human monocytes migrating through the pores of a filter in response to a chemokine diffusing up from beneath（2）细胞增殖或增殖抑制实验可溶性细胞因子检测 （ELISA ） 分泌细胞因子的细胞检测(ELISPOT)胞内蛋白或受体的研究 （免疫组化）免疫印迹法 (Wes