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-82021/7/15 星期四1Just as hormones serve

2、as messenger of 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.2021/7/15 星期四2Cytokines can exert different actionsIL-2Proliferation, survivalExample:Chemokin

3、esLeukocyte recruitmentAcute phase proteins, IL-6TNFIL-332021/7/15 星期四3Properties of cytokines細胞因子調節IgG類別轉換 多效性 拮抗性 重疊性協同性2021/7/15 星期四4Biological effects of the cytokinesAnti-infection and antitumorImmune regulationApoptosisStimulating the proliferation and differentiation of hemopoietic stem cells

4、Promote the growth and differentiation of various cellsParticipation and regulation of the inflammatory responsesCytokine abnormalities in the pathogenesis in certain diseases 2021/7/15 星期四5Cytokines during hematopoiesis2021/7/15 星期四6interleukin, IL （白細胞介素）colony stimulating factor,CSF（集落刺激因子）interf

5、eron,IFN（干擾素）tumor necrosis factor,TNF （腫瘤壞死因子）Chemokine, CK （趨化因子）growth factor,GF （生長因子）Cytokines2021/7/15 星期四7Cytokines of the innate and adaptive immune system2021/7/15 星期四82021/7/15 星期四9Cytokines of innate immune system TNF, IL-1 and IL-6: pleiotropic mediators of inflammation (activators of en

6、dothelium, immune cells, liver and brain)Chemokines: regulators of immune cell adhesion at infection sitesIL-12: inducer of IFN-, increase of cytotoxic activityType I IFN: inducers of antiviral state2021/7/15 星期四10 InterleukinInterleukins are secreted by some leukocytes and act upon other leukocytes

7、, IL-1IL-35 have been identified.2021/7/15 星期四112021/7/15 星期四12 Interleukin-1, IL-12021/7/15 星期四13The release of TNF-a by macrophages induces local protective effects, but TNF-a can have damaging effects when released systemically (septic shock).Tumor necrosis factor,TNFThe cytokines that induce dir

8、ectly apoptosis of tumor cells, including TNF, TNF (Lymphotoxin, LT） , FASL, CD70L, CD30L, CD40L, OX40L, TRAIL (TNF related apoptosis-inducing ligand) 2021/7/15 星期四14 Chemokinechemokines, a group of low-molecular-weight cytokines including interleukin 8, that affect chemotaxis and other aspects of l

9、eukocyte behavior. 2021/7/15 星期四15CX3CRCXCRCX3CCXCCK及CKR分類CCCCCRXCR1999年國際命名委員會根據CK的結構特征命名依據N端前兩個半胱氨酸(Cys)的相對位置不同分為:2021/7/15 星期四162021/7/15 星期四172021/7/15 星期四18IL-12 produced by macrophages and dendritic cells is a key cytokine to fight intracellular microbes (bacteria and viruses)IL-12 induces IFN

10、-, an activator of macrophages (killing of phagocytosed microbes) and Th1 cell differentiationIL-12 also increases cytotoxic activity of CD8 and NK cellsIL-122021/7/15 星期四19The cytokines that resist viral infection, and interfere viral replication, including IFN, IFN, IFN, IFN, IFN ( type I ); IFN (

11、 type II ) type I InterferonAntiviral effects of Type I Interferons (IFN-a/b)2021/7/15 星期四202021/7/15 星期四21Integrative signaling of cytokines during innate immune responses2021/7/15 星期四22Cytokines of adaptive immune systemIL-2: prototypic lymphocyte growth factorIL-4: inducer of Th2 cells, B cell di

12、fferentitation factorIFN-: inducer of Th1 cells2021/7/15 星期四23IL-2 (又稱T細胞生長因子,TCGF) ： 細胞來源：主要由T細胞產生。 作用方式：以自分泌和旁分泌方式發揮效應。 主要生物學功能： （1）活化T細胞，促進細胞因子產生； （2）刺激NK細胞增殖，增強NK殺傷活性及產生 細胞因子；誘導LAK細胞產生 （3）促進B細胞增殖和分泌抗體； （4）激活巨噬細胞。2021/7/15 星期四242021/7/15 星期四25IL-4 induces Isotype switching against extracellular p

13、athogens, such as helminths and athropodesIL-42021/7/15 星期四26Pleiotropic activities 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

14、 on phagocytes strong macrophage activator 2021/7/15 星期四27Anti-inflammatory cytokinesIL-10IL-10: 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 cel

15、ls (Th3) to decrease immune responsesThe TGF receptor is a dimer of ALK and TGF-RII which signal through Smad transcription factorsAbsence of TGF or TGF-RII induces autoimmunity in mice2021/7/15 星期四28Additional key cytokines IL-5: activator of eosinophils, also inducing production of additional eosi

16、nophils from bone marrow progenitorsIL-13: similar to IL-4, but in addition stimulates mucus production and tissue 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 m

17、obilize neutrophils during inflammation (clinical use)2021/7/15 星期四29Colony stimulating factor, CSFThe cytokines that stimulate hemopoietic cells to form cell colony, Participate hemopoietic function are called CSF, for example GM-CSF,G-CSF,M-CSF, Epo,Tpo2021/7/15 星期四30是一類可介導不同類型細胞生長和分化的細胞因子。根據其功能和作

18、用的靶細胞不同，分別命名為：表皮生長因子（epidermal growth factor, EGF)血管內皮生長因子（vascular endothelial growth factor, VEGF)成纖維細胞生長因子（fibroblast growth factor, FGF)神經生長因子(nerve growth factor, NGF)血小板衍生的生長因子（platelet derived growth factor, PDGF) 肝細胞生長因子（hepatic growth factor, HGF)Growth factor2021/7/15 星期四31VEGF Receptor Si

19、gnaling Growth factors applied to angiogenesisVEGF promotes Tumor Angiogenesis2021/7/15 星期四32TH1 and TH2 subsets secrete different cytokinesTH1- cell-mediated functions; inflammationTH2- humoral immunity2021/7/15 星期四33TH1 and TH2 subsets regulate each other 2021/7/15 星期四34CD4+ T cells isolation and

20、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 with anti-mouse CD3 and anti-mouse CD28. rmIL-2 plus anti-mouse IL-4 and anti-mouse IFN-antibodies (T

21、h0 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 anti-mouse CD3-coated plates in the absence or in the presence of anti-mouse CD28 for 24 hours, an

22、d cytokine secretion was determined by ELISA.The red line represents GATA3 staining, the black line corresponds to isotype control. 2021/7/15 星期四352021/7/15 星期四362021/7/15 星期四37細胞因子受體分類主要包括免疫球蛋白受體超家族(1gRSF)、I型細胞因子受體家族、型細胞因子受體家族(干擾素受體家族)、型細胞因子受體家族(腫瘤壞死因子受體家族，TNFRF)和七次跨膜受體家族(又稱G蛋白偶聯受體家族)2021/7/15 星期四3

23、8Cytokine receptors belong to families of receptor proteins, each with a distinctive structure2021/7/15 星期四392021/7/15 星期四402021/7/15 星期四412021/7/15 星期四422021/7/15 星期四432021/7/15 星期四442021/7/15 星期四452021/7/15 星期四46The various members of the interleukin-1 (IL-1) family and their receptors 2021/7/15 星

24、期四472021/7/15 星期四48 趨化因子受體（ GPCRs，G-protein coupled receptors ） 屬于7次跨膜受體超家族，胞內環狀結構與Gi/Go蛋白偶聯胞外細胞膜 細胞質COOHNH2G 蛋白2021/7/15 星期四49Signaling 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 recep

25、tor signaling through IRAKReceptor-associated kinases: TGF- receptors, c-kitG-protein-coupled signaling2021/7/15 星期四50Class I and Class II cytokine receptors signal by a rapid pathway using receptor-associated kinases to activate specific transcription factors.Jak-STAT signaling pathways, common to

26、many cytokine receptors4 Jak proteins known: Jak1-3 and Tyk26 STATs known: STAT1-4, 5a, 5b and 6Receptor dimerization central for signalingCombination of Jak/STATs confers specificity2021/7/15 星期四51JAK/STATs Used by Key CytokinesIL-5-Eosinophil2021/7/15 星期四52IL-1TNFGM-CSFM-CSFFGFPDGFVEGFIL-12IL-15IL

27、-6LIFOSMChemokinesTGFIL-10IL-11IL-13sTNF-RIL-1raPRO-INFLAMMATORYANTI-INFLAMMATORYCytokine imbalance during inflammation2021/7/15 星期四532021/7/15 星期四54 CXCR4（T細胞）與CCR5（巨噬細胞） 是HIV感染靶細胞的共受體。2021/7/15 星期四55細胞因子抑制劑 用于治療炎癥，自身免疫病，移植排斥，休克細胞因子的臨床應用2021/7/15 星期四56進入臨床實驗的細胞因子抑制劑名稱公司適應癥臨床階段可溶性IL-1受體(干粉吸入劑)Immune

28、x哮喘I/II期可溶性IL-1受體（注射劑）急性髓樣白血病I期IL-1受體拮抗劑Amegen敗血性休克(試驗中止),類風濕關節炎完成III期可溶性IL-4受體Immunex哮喘I/II期抗IL-4人源化抗體SmithKline哮喘I/II期抗IL-5人源化抗體SmithKline哮喘II期可溶性TNF受體II-Fc融合蛋白Immunex類風濕關節炎，慢性心衰上市，I期可溶性TNF受體I-Fc融合蛋白Hoffmann-La Roch休克,類風濕關節炎,多發性硬化癥II/III期抗TNF單抗（infliximab）CentocorCrohns Disease上市人源化抗HER2 (EGFR2)單抗

29、Genentech乳腺癌轉移上市 DAB389-IL-2(IL-2免疫毒素)SeragenT細胞淋巴瘤, I型糖尿病,嚴重類風濕關節炎,牛皮癬,HIV感染上市I/II期2021/7/15 星期四57DAB389-EGFSeragen腫瘤I/II期人源化抗VEGF單抗Genentech Inc.晚期肺癌，腸癌III期抗VEGFR2（KDR）嵌合抗體Albert LoBuglio轉移性腸癌I期人源化抗IL-8抗體（ABX-IL8）AbgenixSevere psoriasisI/II期臨床IL13-PE38QQR（IL-13免疫毒素）NeoPharm, Inc腎癌I期臨床TNF反義寡核苷酸Isis

30、類風濕關節炎，牛皮癬I期臨床抗eotaxin抗體（CAT-213）Cambridge Antibody變態反應I期臨床2021/7/15 星期四58IL-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 clone vector2021/7/15 星期四59Subcloneeukar

31、yotic expression vectorspcI-neoEcoRIEcoRIEcoRICIPEcoRI+ligationpcI-IL-1raEcoRIEcoRIidentifiedPCRRestriction enzyme2021/7/15 星期四60pcDI-IL-1ratransfectionsynoviocyteCOS-7in vitroEXPRESSIONIL-1ra proteinELISAmRNA2021/7/15 星期四61pcDI-IL-1rain vivo (gene therapy)muscle injection.DBA/1 micetype II collagen

32、inducedDBA/1 mice with arthritis gene gun2021/7/15 星期四62細胞因子的臨床應用2021/7/15 星期四63 已批準上市的細胞因子基因工程藥物名稱適應癥IFN白血病、Kaposi肉瘤、肝炎、癌癥、AIDSIFN慢性肉芽腫、生殖器疣、過敏性皮炎、感染性疾病、類風濕關節炎G-CSF自身骨髓移植、化療導致的粒細胞減少癥、 AIDS、白血病、再生障礙性貧血GM-CSF自身骨髓移植、化療導致的血細胞減少癥、 AIDS、再生障礙性貧血、Epo慢性腎衰導致的貧血、癌癥或癌癥化療導致的貧血、失血后貧血IL-2癌癥、免疫缺陷、疫苗佐劑IFN多發性硬化癥IL-1

33、1放化療所致血小板減少癥干細胞因子(SCF)與G-CSF聯合應用于外周血干細胞移植EGF外用藥治療燒傷、潰瘍bFGF外用藥治療燒傷、外周神經炎2021/7/15 星期四64正進行臨床試驗的細胞因子基因工程藥物細胞因子名稱適應癥公司IL-10炎癥,銀屑病,Crohns 病,類風濕關節炎Immunex，Schering-PloughIL-12腫瘤，HIV感染，I型變態反應 GITpo血小板減少癥GI，沈陽三生TNF變異體腫瘤國內多家企業Flt3/flk3 Ligand(FL)腫瘤ImmunexMyeloid Progenitor Inhibitory Factor-1 (MPIF-1)腫瘤大劑量化

34、療HGSTGF-慢性皮膚潰瘍，多發性硬化癥GenzymeKeratinocyte Growth Factor-2 (KGF-2)促進燒傷、慢性潰瘍的傷口愈合；抗癌藥物引起的粘膜損傷；炎癥性腸道疾病HGSMIP-1 變異體腫瘤化療的骨髓保護作用British BiotechOPG（osteoprotegerin）骨質疏松AmgenMulitkine（白細胞產生的細胞因子混合制劑）轉移性腫瘤CEL-SCIB細胞刺激因子（BlyS）免疫缺陷HGS2021/7/15 星期四65二、細胞因子的研究熱點1、新細胞因子的基因克隆2、細胞因子受體的基因克隆3、細胞因子信號轉導機制4、新一代細胞因子：高活性，多

35、功能，低毒副作用，長半衰期，高穩定性5、細胞因子作為生物應答調節劑（BRM）的臨床應用6、細胞因子表達調控7、細胞因子基因治療2021/7/15 星期四661、新細胞因子的基因克隆化從人類基因組發現新細胞因子后基因組時代的“撈魚”2021/7/15 星期四672、可溶性細胞因子受體作為拮抗劑effectsNC-S-S-S-S-S-S-N-S-S-S-S-2021/7/15 星期四68NC53DNAProtein（1）、細胞因子受體的克隆NC-S-S-S-S-S-S-2021/7/15 星期四6953DNAPCR可溶性細胞因子受體expressN-S-S-S-S-2021/7/15 星期四70R

36、eceptor 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-2021/7/15 星期四71（2）、細胞因子受體的突變CN5353TAASite-directed mutagenesisSoluble cytokine receptorCN2021/7/15 星期四72細胞因子的檢測1 生物學活性檢測2 免疫學檢測3 分子生物學檢測2021/7/15 星期四731 生物學活性檢測（1）趨化因子的檢測2021/7/15 星期四74顯微鏡下觀察, 計數 : 細胞數/

37、視野2021/7/15 星期四75This scanning electron micrograph shows two human monocytes migrating through the pores of a filter in response to a chemokine diffusing up from beneath2021/7/15 星期四76（2）細胞增殖或增殖抑制實驗2021/7/15 星期四77可溶性細胞因子檢測 （ELISA ） 分泌細胞因子的細胞檢測(ELISPOT)胞內蛋白或受體的研究 （免疫組化）免疫印跡法 (Western blotting) 細胞因子或受體（抗原）+（人）特異性單