2022醫(yī)學(xué)課件博士生課程5(1)-細(xì)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-8第一頁，共八十九頁。Just as hormones serve as m

2、essenger 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.第二頁，共八十九頁。Cytokines can exert different actionsIL-2Proliferation, survivalExample:ChemokinesLeukoc

3、yte recruitmentAcute phase proteins, IL-6TNFIL-33第三頁，共八十九頁。Properties of cytokines細(xì)胞因子調(diào)節(jié)IgG類別轉(zhuǎn)換 多效性 拮抗性 重疊性協(xié)同性第四頁，共八十九頁。Biological effects of the cytokinesAnti-infection and antitumorImmune regulationApoptosisStimulating the proliferation and differentiation of hemopoietic stem cellsPromote the grow

4、th and differentiation of various cellsParticipation and regulation of the inflammatory responsesCytokine abnormalities in the pathogenesis in certain diseases 第五頁，共八十九頁。Cytokines during hematopoiesis第六頁，共八十九頁。interleukin, IL 白細(xì)胞介素colony stimulating factor,CSF集落刺激因子interferon,IFN干擾素tumor necrosis fa

5、ctor,TNF 腫瘤壞死因子Chemokine, CK 趨化因子growth factor,GF 生長因子Cytokines第七頁，共八十九頁。Cytokines of the innate and adaptive immune system第八頁，共八十九頁。第九頁，共八十九頁。Cytokines of innate immune system TNF, IL-1 and IL-6: pleiotropic mediators of inflammation (activators of endothelium, immune cells, liver and brain)Chemoki

6、nes: regulators of immune cell adhesion at infection sitesIL-12: inducer of IFN-, increase of cytotoxic activityType I IFN: inducers of antiviral state第十頁，共八十九頁。 InterleukinInterleukins are secreted by some leukocytes and act upon other leukocytes, IL-1IL-35 have been identified.第十一頁，共八十九頁。第十二頁，共八十九

7、頁。 Interleukin-1, IL-1第十三頁，共八十九頁。The 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 directly apoptosis of tumor cells, including TNF, TNF (Lymphotoxin,

8、LT , FASL, CD70L, CD30L, CD40L, OX40L, TRAIL (TNF related apoptosis-inducing ligand) 第十四頁，共八十九頁。 Chemokinechemokines, a group of low-molecular-weight cytokines including interleukin 8, that affect chemotaxis and other aspects of leukocyte behavior. 第十五頁，共八十九頁。CX3CRCXCRCX3CCXCCK及CKR分類CCCCCRXCR1999年國際

9、命名委員會根據(jù)CK的結(jié)構(gòu)特征命名依據(jù)N端前兩個半胱氨酸(Cys)的相對位置不同分為:第十六頁，共八十九頁。第十七頁，共八十九頁。第十八頁，共八十九頁。IL-12 produced by macrophages and dendritic cells is a key cytokine to fight intracellular microbes (bacteria and viruses)IL-12 induces IFN-, an activator of macrophages (killing of phagocytosed microbes) and Th1 cell differe

10、ntiationIL-12 also increases cytotoxic activity of CD8 and NK cellsIL-12第十九頁，共八十九頁。The cytokines that resist viral infection, and interfere viral replication, including IFN, IFN, IFN, IFN, IFN ( type I ); IFN ( type II ) type I InterferonAntiviral effects of Type I Interferons (IFN-a/b)第二十頁，共八十九頁。第二

11、十一頁，共八十九頁。Integrative signaling of cytokines during innate immune responses第二十二頁，共八十九頁。Cytokines of adaptive immune systemIL-2: prototypic lymphocyte growth factorIL-4: inducer of Th2 cells, B cell differentitation factorIFN-: inducer of Th1 cells第二十三頁，共八十九頁。IL-2 (又稱T細(xì)胞生長因子,TCGF) ： 細(xì)胞來源：主要由T細(xì)胞產(chǎn)生。 作用

12、方式：以自分泌和旁分泌方式發(fā)揮效應(yīng)。 主要生物學(xué)功能： 1活化T細(xì)胞，促進(jìn)細(xì)胞因子產(chǎn)生； 2刺激NK細(xì)胞增殖，增強(qiáng)NK殺傷活性及產(chǎn)生 細(xì)胞因子；誘導(dǎo)LAK細(xì)胞產(chǎn)生 3促進(jìn)B細(xì)胞增殖和分泌抗體； 4激活巨噬細(xì)胞。第二十四頁，共八十九頁。第二十五頁，共八十九頁。IL-4 induces Isotype switching against extracellular pathogens, such as helminths and athropodesIL-4第二十六頁，共八十九頁。Pleiotropic activities of IFN-IFN- is secreted by activated

13、 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 第二十七頁，共八十九頁。Anti-inflammatory cytokinesIL-10IL-10: counteracts IFN- by blocki

14、ng 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 through Smad transcription

15、 factorsAbsence of TGF or TGF-RII induces autoimmunity in mice第二十八頁，共八十九頁。Additional 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 tissue fibrosis in

16、 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)第二十九頁，共八十九頁。Colony stimulating factor, CSFThe cytokines that stimulate h

17、emopoietic cells to form cell colony, Participate hemopoietic function are called CSF, for example GM-CSF,G-CSF,M-CSF, Epo,Tpo第三十頁，共八十九頁。是一類可介導(dǎo)不同類型細(xì)胞生長和分化的細(xì)胞因子。根據(jù)其功能和作用的靶細(xì)胞不同，分別命名為：表皮生長因子epidermal growth factor, EGF)血管內(nèi)皮生長因子vascular endothelial growth factor, VEGF)成纖維細(xì)胞生長因子fibroblast growth factor,

18、FGF)神經(jīng)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(xì)胞生長因子hepatic growth factor, HGF)Growth factor第三十一頁，共八十九頁。VEGF Receptor Signaling Growth factors applied to angiogenesisVEGF promotes Tumor Angiogenesis第三十二頁，共八十九頁。TH1 and TH2 subsets secrete different cytokinesTH1-

19、 cell-mediated functions; inflammationTH2- humoral immunity第三十三頁，共八十九頁。TH1 and TH2 subsets regulate each other 第三十四頁，共八十九頁。CD4+ T cells isolation and differentiationSpleens were mechanically dissociated, followed by depletion of erythrocytes. Naive CD4+ T cells were isolated by negative selection. C

20、D4+ 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 (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

21、. 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, and cytokine secretion was determined by ELISA.The red line represents GATA3 staining, the black line corresponds to isotype control. 第三十五頁，共八十九頁。第三十六頁，

22、共八十九頁。第三十七頁，共八十九頁。細(xì)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(xì)胞因子受體家族、型細(xì)胞因子受體家族(干擾素受體家族)、型細(xì)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(lián)受體家族)第三十八頁，共八十九頁。Cytokine receptors belong to families of receptor proteins, each with a distinctive structure第三十九頁，共八十九頁。第四十頁，共八十九頁。第四十一頁，共八十九頁。第四十二頁，共八十九頁。第四十三頁，共八十九頁。第四十四頁，共八十

23、九頁。第四十五頁，共八十九頁。第四十六頁，共八十九頁。The various members of the interleukin-1 (IL-1) family and their receptors 第四十七頁，共八十九頁。第四十八頁，共八十九頁。 趨化因子受體 GPCRs，G-protein coupled receptors 屬于7次跨膜受體超家族，胞內(nèi)環(huán)狀結(jié)構(gòu)與Gi/Go蛋白偶聯(lián)胞外細(xì)胞膜 細(xì)胞質(zhì)COOHNH2G 蛋白第四十九頁，共八十九頁。Signaling of cytokine receptorsType I/II cytokine receptors: Jak-STAT pa

24、thwayTNF 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 signaling第五十頁，共八十九頁。Class I and Class II cytokine receptors signal by a rapid pathway using receptor-asso

25、ciated 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 signalingCombination of Jak/STATs confers specificity第五十一頁，共八十九頁。JAK/STAT

26、s Used by Key CytokinesIL-5-Eosinophil第五十二頁，共八十九頁。IL-1TNFGM-CSFM-CSFFGFPDGFVEGFIL-12IL-15IL-6LIFOSMChemokinesTGFIL-10IL-11IL-13sTNF-RIL-1raPRO-INFLAMMATORYANTI-INFLAMMATORYCytokine imbalance during inflammation第五十三頁，共八十九頁。第五十四頁，共八十九頁。 CXCR4T細(xì)胞與CCR5巨噬細(xì)胞 是HIV感染靶細(xì)胞的共受體。第五十五頁，共八十九頁。細(xì)胞因子抑制劑 用于治療炎癥，自身免疫病，

27、移植排斥，休克細(xì)胞因子的臨床應(yīng)用第五十六頁，共八十九頁。進(jìn)入臨床實(shí)驗(yàn)的細(xì)胞因子抑制劑名稱公司適應(yīng)癥臨床階段可溶性IL-1受體(干粉吸入劑)Immunex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(yàn)中止),類風(fēng)濕關(guān)節(jié)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(fēng)濕關(guān)節(jié)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(fēng)濕關(guān)節(jié)炎,

28、多發(fā)性硬化癥II/III期抗TNF單抗infliximabCentocorCrohns Disease上市人源化抗HER2 (EGFR2)單抗Genentech乳腺癌轉(zhuǎn)移上市 DAB389-IL-2(IL-2免疫毒素)SeragenT細(xì)胞淋巴瘤, I型糖尿病,嚴(yán)重類風(fēng)濕關(guān)節(jié)炎,牛皮癬,HIV感染上市I/II期第五十七頁，共八十九頁。DAB389-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(zhuǎn)移性腸癌I期人源化抗IL-8抗體（ABX-IL8）AbgenixSevere pso

29、riasisI/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類風(fēng)濕關(guān)節(jié)炎，牛皮癬I期臨床抗eotaxin抗體（CAT-213）Cambridge Antibody變態(tài)反應(yīng)I期臨床第五十八頁，共八十九頁。IL-1ra gene therapy on DBA/1 mice of collagen-induced arthritis cDNA librariesPCRTTTTTTTTAA+sequencingIL-1ra geneligateIL-1ra: IL-1 receptor antagonistAT clone s

30、ystem (Invitrogen Co.)AT clone vector第五十九頁，共八十九頁。Subcloneeukaryotic expression vectorspcI-neoEcoRIEcoRIEcoRICIPEcoRI+ligationpcI-IL-1raEcoRIEcoRIidentifiedPCRRestriction enzyme第六十頁，共八十九頁。pcDI-IL-1ratransfectionsynoviocyteCOS-7in vitroEXPRESSIONIL-1ra proteinELISAmRNA第六十一頁，共八十九頁。pcDI-IL-1rain vivo (g

31、ene therapy)muscle injection.DBA/1 micetype II collageninducedDBA/1 mice with arthritis gene gun第六十二頁，共八十九頁。細(xì)胞因子的臨床應(yīng)用第六十三頁，共八十九頁。 已批準(zhǔn)上市的細(xì)胞因子基因工程藥物名稱適應(yīng)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(fēng)濕關(guān)節(jié)炎G-CSF自身骨髓移植、化療導(dǎo)致的粒細(xì)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(dǎo)致的血細(xì)胞減少癥、 AIDS、再生障礙性貧血、Epo慢性腎衰導(dǎo)致的貧

32、血、癌癥或癌癥化療導(dǎo)致的貧血、失血后貧血IL-2癌癥、免疫缺陷、疫苗佐劑IFN多發(fā)性硬化癥IL-11放化療所致血小板減少癥干細(xì)胞因子(SCF)與G-CSF聯(lián)合應(yīng)用于外周血干細(xì)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(jīng)炎第六十四頁，共八十九頁。正進(jìn)行臨床試驗(yàn)的細(xì)胞因子基因工程藥物細(xì)胞因子名稱適應(yīng)癥公司IL-10炎癥,銀屑病,Crohns 病,類風(fēng)濕關(guān)節(jié)炎Immunex，Schering-PloughIL-12腫瘤，HIV感染，I型變態(tài)反響 GITpo血小板減少癥GI，沈陽三生TNF變異體腫瘤國內(nèi)多家企業(yè)Flt3/flk3 Ligand(FL)腫瘤ImmunexMyeloid

33、 Progenitor Inhibitory Factor-1 (MPIF-1)腫瘤大劑量化療HGSTGF-慢性皮膚潰瘍，多發(fā)性硬化癥GenzymeKeratinocyte Growth Factor-2 (KGF-2)促進(jìn)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(hù)作用British BiotechOPGosteoprotegerin骨質(zhì)疏松AmgenMulitkine白細(xì)胞產(chǎn)生的細(xì)胞因子混合制劑轉(zhuǎn)移性腫瘤CEL-SCIB細(xì)胞刺激因子BlyS免疫缺陷HGS第六十五頁，共八十九頁。二、細(xì)胞因子的研究熱點(diǎn)1、新細(xì)胞因子的基因克隆2、細(xì)

34、胞因子受體的基因克隆3、細(xì)胞因子信號轉(zhuǎn)導(dǎo)機(jī)制4、新一代細(xì)胞因子：高活性，多功能，低毒副作用，長半衰期，高穩(wěn)定性5、細(xì)胞因子作為生物應(yīng)答調(diào)節(jié)劑BRM的臨床應(yīng)用6、細(xì)胞因子表達(dá)調(diào)控7、細(xì)胞因子基因治療第六十六頁，共八十九頁。1、新細(xì)胞因子的基因克隆化從人類基因組發(fā)現(xiàn)新細(xì)胞因子后基因組時代的“撈魚第六十七頁，共八十九頁。2、可溶性細(xì)胞因子受體作為拮抗劑effectsNC-S-S-S-S-S-S-N-S-S-S-S-第六十八頁，共八十九頁。NC53DNAProtein1、細(xì)胞因子受體的克隆NC-S-S-S-S-S-S-第六十九頁，共八十九頁。53DNAPCR可溶性細(xì)胞因子受體expressN-S-S-

35、S-S-第七十頁，共八十九頁。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-第七十一頁，共八十九頁。2、細(xì)胞因子受體的突變CN5353TAASite-directed mutagenesisSoluble cytokine receptorCN第七十二頁，共八十九頁。細(xì)胞因子的檢測1 生物學(xué)活性檢測2 免疫學(xué)檢測3 分子生物學(xué)檢測第七十三頁，共八十九頁。1 生物學(xué)活性檢測1趨化因子的檢測第七十四頁，共八十九頁。顯微鏡下觀察, 計數(shù) : 細(xì)胞數(shù)/

36、視野第七十五頁，共八十九頁。This scanning electron micrograph shows two human monocytes migrating through the pores of a filter in response to a chemokine diffusing up from beneath第七十六頁，共八十九頁。2細(xì)胞增殖或增殖抑制實(shí)驗(yàn)第七十七頁，共八十九頁?？扇苄约?xì)胞因子檢測 ELISA 分泌細(xì)胞因子的細(xì)胞檢測(ELISPOT)胞內(nèi)蛋白或受體的研究 免疫組化免疫印跡法 (Western blotting) 細(xì)胞因子或受體抗原+人特異性單克隆抗體一抗+酶標(biāo)的兔羊抗人抗體二抗2 免疫學(xué)檢測法檢測細(xì)胞因子蛋白質(zhì)的抗原特性第七十八頁，共八十九頁。ELISA第七十九頁，共八十九頁。Colorimetric ELISA Substrates 第八十頁，共八十九頁。ELISPOT第八十一頁，共八十九頁。第八十二頁，共八十九頁。ABCDEFGHIJKL1POSPOSNEGNEGBlankBLCCD30 LEotax