摘要
论文以模式生物斑马鱼作为研究对象,开展了鱼类白介素-4(interleukin-4,IL-4)及其受体IL-4Rα的分子克隆与功能研究。
论文第一部分为绪论部分。主要分两个部分介绍了IL-4及其特异性受体IL-4Rα的研究近况。第一部分介绍了IL-4的发现、基因克隆、基因结构、表达调控机制、受体的组成、生物学功能以及相关信号传导途径等方面的研究结果和进展;第二部分主要介绍了IL-4Rα的基因克隆、表达调控、信号传导、选择剪接形式及生物学功能等方面的研究进展。IL-4Rα和IL-4是高度亲和的一对特异性相互作用的分子,两者的亲和作用对发挥IL-4的生物学功能起到了非常重要的作用。
论文第二部分为实验部分。主要分为三个小节:第一小节,为斑马鱼IL-4基因的克隆、生物信息学分析以及组织分布的鉴定,同时利用原核表达系统得到可溶型的重组斑马鱼IL-4蛋白质。利用生物信息学软件,分析了斑马鱼IL-4分子在进化上的规律,并且分析了其在进化上与高等脊椎动物及其他物种的关系。第二部分,在斑马鱼中克隆得到了IL-4特异性受体IL-4Rα的基因,同时还克隆到一个IL-4Rα样(IL-4Rα-L)的基因,与IL-4Rα基因座位相邻,它与IL-4Rα基因在序列和结构上存在高度的同源性,最大的区别是IL-4Rα-L缺失大部分的胞内区,同时两者通过选择性剪接均得到一个可溶形式的蛋白。我们对这四个分子进行了其在斑马鱼中组织分布的规律的研究,同时在原核表达系统中表达并纯化得到可溶性的IL-4Rα蛋白质,并制备得到多克隆抗体。利用生物信息学软件,对IL-4Rα及其IL-4Rα-L与其他物种中的IL-4Rα进行分析比对,发现IL-4Rα在基因结构,基因共线性,蛋白质的空间构象,蛋白质信号转导的结合位点等各方面都是相当保守的。第三部分,我们对IL-4/IL-4Rα进行了功能分析,提供了IL-4和IL-4Rα作为一对特异性互作分子的证据,同时发现IL-4能刺激B细胞增殖,对抗体产生起上调作用,并且鉴定了IL-4提高Th2类细胞因子及转录因子的表达,证实了IL-4/IL-4Rα作为Th2类免疫调节中一个重要的信号调节途径在鱼类中的发生。
This article focused on the molecular cloning, functional characterization of Th2 immunity related molecules and preliminary identification of Th2 immunity in Danio rerio.
Chapter 1 is an introduction of the recent researches on IL-4 and its specific receptor IL-4Rα. It was described in two parts. In the first part, we mainly introduce interleukin-4 (IL-4) and IL-4Ra (IL-4 receptor alpha chain) gene including its discovery, molecular cloning, gene structure, regulatory mechanisms of them expression, its receptors and the corresponding signal pathways, the biology functions of IL-4, as well as various types of alternative splicing of IL-4Rα. IL-4 and IL-4Ra was two reciprocal molecules with high affinity, they play the important function in Th2 immunity.
Chapter 2 is the description of the main researches. It was described in three parts. In first part, we performed the molecular cloning and characterization of IL-4 from zebrafish, and its prokaryotic expression in E.coli and purification of soluble recombinant protein. By bioinformatics methods, we carried out the study on the evolution of vertebrates IL-4 genes. In the second part, we cloned IL-4Rα, and we also get a IL-4Rαlike (IL-4Rα-L) gene from zebrafish. IL-4Ra-L adjacent to IL-4Ra, they had high identity in cDNA sequences and gene structures, the most difference is that IL-4Rα-L laking the most cytoplasmic region. Both of them have its solubles from alternative splicing. By bioinformatics methods, we found that the gene structure, gene locus, protein structure and the main motifs for signaling were conserved across all species aligned. In the third part, we performed the function analysis on IL-4/IL-4Rα, provided the evidence on the two reciprocal molecules. And we found that, IL-4 could stimulate the proliferation of B cell, up-regulate the production of antibody, and promote the expression of Th2 cytokines and transcription factors. All the evidence showed that a possible Th2-mediated regulatory mechanism had been developed in the adaptive immunity during early vertebrate evolution.
引文
1.Bazan,J.F.,Haemopoietic receptors and helical cytokines.Immunol Today,1990.11(10):p.350-4.
2.Grabstein,K.H.,et al.,Cloning of a T cell growth factor that interacts with the beta chain of the interleukin-2 receptor.Science,1994.264(5161):p.965-8.
3.Gustchina,A.,et al.,A model of the complex between interleukin-4 and its receptors.Proteins,1995.21(2):p.140-8.
4.Farrar,J.D.,H.Asnagli,and K.M.Murphy,T helper subset development:roles of instruction,selection,and transcription.J Clin Invest,2002.109(4):p.431-5.
5.Pancer,Z.and M.D.Cooper,The evolution of adaptive immunity.Annu Rev Immunol,2006.24:p.497-518.
6.Howard,M.and W.E.Paul,Interleukinsfor B lymphocytes.Lymphokine Res,1982.1(1):p.1-4.
7.Vitetta,E.S.,et al.,Serological,biochemical and functional identity of B cell-stimulatory factor 1 and B cell differentiation factor for IgG1.J Exp Med,1985.162(5):p.1726-31.
8.Yokota,T.,et al.,Isolation and characterization of a human interleukin cDNA clone,homologous to mouse B-cell stimulatory factor 1,that expresses B-cell-and T-cell-stimulating activities.Proc Natl Acad Sci U S A,1986.83(16):p.5894-8.
9.Le,H.V.,et al.,Isolation and characterization of multiple variants of recombinant human interleukin 4 expressed in mammalian cells.J Biol Chem,1988.263(22):p.10817-23.
10.Smith,L.J.,et al.,Human interleukin 4.The solution structure of a four-helix bundle protein.J Mol Biol,1992.224(4):p.899-904.
11.Jelinek,D.F.,Regulation of B lymphocyte differentiation.Ann Allergy Asthma Immunol,2000.84(4):p.375-85;quiz 385-7.
12.Noelle,R.and E.C.Snow,Thelper cells.Curr Opin Immunol,1992.4(3):p.333-7.
13.Li,J.H.,et al.,Cloning,characterization and expression analysis of pufferfish interleukin-4cDNA:the first evidence of Th2-type cytokine in fish.Mol Immunol,2007.44(8):p.2078-86.
14.Seder,R.A.and W.E.Paul,Acquisition of lymphokine-producing phenotype by CD4+ T cells.Annu Rev Immunol,1994.12:p.635-73.
15.Del Prete,G.,M.Ricci,and S.Romagnani,T cells,IgE antibodies,cytokines and allergic inflammation.Allerg Immunol(Paris),1991.23(6):p.239-43.
16.Mosmann,T.R.and R.L.Coffman,TH1 and TH2 cells:different patterns of lymphokine secretion lead to different functional properties.Annu Rev Immunol,1989.7:p.145-73.
17.Ferrick,D.A.,et al.,Differential production of interferon-gamma and interleukin-4 in response to Th1-and Th2-stimulating pathogens by gamma delta T cells in vivo.Nature,1995.373(6511):p.255-7.
18.Barner,M.,et al.,Differences between IL-4R alpha-defcient and IL-4-deficient mice reveal a role for IL-13 in the regulation of Th2 responses.Curr Biol,1998.8(11):p.669-72.
19.Chen,Z.,et al.,Identification of novel IL-4/Stat6-regulated genes in T lymphocytes.J Immunol,2003.171(7):p.3627-35.
20.Min,B.and W.E.Paul,Basophils and type 2 immunity.Curr Opin Hematol,2008.15(1):p.59-63.
21.Plaut,M.,et al.,Mast cell lines produce lymphokines in response to cross-linkage of Fc epsilon RI or to calcium ionophores.Nature,1989.339(6219):p.64-7.
22.Solymar,D.C.,et al.,A 3' enhancer in the IL-4 gene regulates cytokine production by Th2cells and mast cells.Immunity,2002.17(1):p.41-50.
23.Kuhn,R.,K.Rajewsky,and W.Muller,Generation and analysis of interleukin-4 deficient mice.Science,1991.254(5032):p.707-10.
24.Shimoda,K.,et al.,Lack of IL-4-induced Th2 response and IgE class switching in mice with disrupted Stat6 gene.Nature,1996.380(6575):p.630-3.
25.Vercelli,D.,et al.,IL-4 inhibits the synthesis of IFN-gamma and induces the synthesis of IgE in human mixed lymphocyte cultures.J Immunol,1990.144(2):p.570-3.
26.Fang,W.,et al,,Identification and characterization of an interleukin-15 homologue from Tetraodon nigroviridis.Comp Biochem Physiol B Bioehem Mol Biol,2006.143(3):p.335-43.
27.Carson,W.E.,et al.,Interleukin(IL) 15 is a novel cytokine that activates human natural killer cells via components of the IL-2 receptor.J Exp Med,1994.180(4):p.1395-403.
28.Giri,J.G.,et al.,Utilization of the beta and gamma chains of the IL-2 receptor by the novel cytokine IL-15.EMBO J,1994.13(12):p.2822-30.
29.Ozaki,K.and W.J.Leonard,Cytokine and cytokine receptor pleiotropy and redundancy.J Biol Chem,2002.277(33):p.29355-8.
30.Nelms,K.,et al.,The IL-4 receptor:signaling mechanisms and biologic functions.Annu Rev Immunol,1999.17:p.701-38.
31.Keegan,A.D.,et al.,The IL-4 receptor:biochemical characterization of IL-4-binding molecules in a T cell line expressing large numbers of receptors.J Immunol,1991.146(7):p.2272-9.
32.Zurawski,S.M.,et al.,Receptors for interleukin-13 and interleukin-4 are complex and share a novel component that functions in signal transduction.EMBO J,1993.12(7):p.2663-70.
33.Guo,J.,et al.,Chromosome mapping and expression of the human interleukin-13 receptor.Genomics,1997.42(1):p.141-5.
34.de Vries,J.E.,The role of IL-13 and its receptor in allergy and inflammatory responses.J Allergy Clin Immunol,1998.102(2):p.165-9.
35.Andrews,A.L.,et al.,Kinetic analysis of the interleukin-13 receptor complex.J Biol Chem,2002.277(48):p.46073-8.
36.Cortes,J.R.,et al.,Kaempferol inhibits IL-4-induced STAT6 activation by specifically targeting JAK3.J Immunol,2007.179(6):p.3881-7.
37.Takeda,K.,et al.,Essential role of Stat6 in IL-4 signalling.Nature,1996.380(6575):p.627-30.
38.Metwali,A.,et al.,Interleukin-4 receptor alpha chain and STAT6 signaling inhibit gamma interferon but not Th2 cytokine expression within schistosome granulomas.Infect Immun,2002.70(10):p.5651-8.
39.Kapsenberg,M.L.,et al.,Role of type 1 and type 2 T helper cells in allergic diseases.Curr Opin Immunol,1992.4(6):p.788-93.
40.Chatila,T.A.,Interleukin-4 receptor signaling pathways in asthma pathogenesis.Trends Mol Med,2004.10(10):p.493-9.
41.Hoffjan,S.and C.Ober,Present status on the genetic studies of asthma.Curr Opin Immunol,2002.14(6):p.709-17.
42.Risma,K.A.,et al.,V75R576 IL-4 receptor alpha is associated with allergic asthma and enhanced IL-4 receptor function.J Immunol,2002.169(3):p.1604-10.
43.Munitz,A.,et al.,Distinct roles for IL-13 and IL-4 via IL-13 receptor alpha1 and the type ⅡIL-4 receptor in asthma pathogenesis.Proc Natl Acad Sci U S A,2008.105(20):p.7240-5.
44.Kips,J.C.,Cytokines in asthma.Eur Respir J Suppl,2001.34:p.24s-33s.
45.Lee,S.O.,et al.,Interleukin-4 stimulates androgen-independent growth in LNCaP human prostate cancer cells.Prostate,2008.68(1):p.85-91.
46.Hill,T.A.,et al.,Intracellular cytokine profiles and T cell activation in pulmonary sarcoidosis.Cytokine,2008.42(3):p.289-92.
47.Kruse,S.,et al.,Characterization of the membrane-bound and a soluble form of human IL-4receptor alpha produced by alternative splicing.Int Immunol,1999.11(12):p.1965-70.
48.Jacobs,C.A.,et al.,Characterization and pharmacokinetic parameters of recombinant soluble interleukin-4 receptor.Blood,1991.77(11):p.2396-403.
49.Galizzi,J.P.,et al.,Molecular cloning of a cDNA encoding the human interleukin 4 receptor.Int Immunol,1990.2(7):p.669-75.
50.Bazan,J.F.,Structural design and molecular evolution of a cytokine receptor superfamily.Proc Natl Acad Sci U S A,1990.87(18):p.6934-8.
51.Wrighton,N.,et al.,The murine interleukin-4 receptor gene:genomic structure,expression and potential for alternative splicing.Growth Factors,1992.6(2):p.103-18.
52.Hage,T.,W.Sebald,and P.Reinemer,Crystal structure of the interleukin-4/receptor alpha chain complex reveals a mosaic binding interface.Cell,1999.97(2):p.271-81.
53.Pan,P.Y.and P.Rothman,IL-4 receptor mutations.Curr Opin Immunol,1999.11(6):p.615-20.
54.Ihle,J.N.,The Janus protein tyrosine kinase family and its role in cytokine signaling.Adv Immunol,1995.60:p.1-35.
55.Jiang,H.,M.B.Harris,and P.Rothman,IL-4/IL-13 signaling beyond JAK/STAT.J Allergy Clin Immunol,2000.105(6 Pt 1):p.1063-70.
56.Liao,W.,et al.,Priming for T helper type 2 differentiation by interleukin 2-mediated induction of interleukin 4 receptor alpha-chain expression.Nat Immunol,2008.9(11):p.1288-96.
57.Duschl,A.and W.Sebald,Transmembrane and intracellular signalling by interleukin-4:receptor dimerization and beyond.Eur Cytokine Netw,1996.7(1):p.37-49.
58.Jung,T.,et al.,Soluble human interleukin-4 receptor is produced by activated T cells under the control of metalloproteinases.Int Arch Allergy Immunol,1999.119(1):p.23-30.
59.Maliszewski,C.R.,et al.,Delayed allograft rejection in mice transgenic for a soluble form of the IL-4 receptor.Cell Immunol,1992.143(2):p.434-48.
60.Steinke,J.W.and L.Borish,Th2 cytokines and asthma,Interleukin-4:its role in the pathogenesis of asthma,and targeting it for asthma treatment with interleukin-4 receptor antagonists.Respir Res,2001.2(2):p.66-70.
61.Cosman,D.,et al.,A new cytokine receptor superfamily.Trends Biochem Sci,1990.15(7):p.265-70.
62.Maliszewski,C.R.,et al.,In vivo biological effects of recombinant soluble interleukin-4receptor.Proc Soc Exp Biol Med,1994.206(3):p.233-7.
63.Puccetti,P.,et al.,Cure of murine candidiasis by recombinant soluble interleukin-4 receptor.J Infect Dis,1994.169(6):p.1325-31.
64.Jacobs,C.A.,et al.,Pharmacokinetic parameters and biodistribution of soluble cytokine receptors.Int Rev Exp Pathol,1993.34 Pt B:p.123-35.
65.Sato,T.A.,et al.,Recombinant soluble murine IL-4 receptor can inhibit or enhance IgE responses in vivo.J Immunol,1993.150(7):p.2717-23.
66.Pollock,H.W.,et al.,The role of soluble interleukin-4 receptor and interleukin-5 antibody in preventing late-phase allergy-induced eustachian tube dysfunction.Otolaryngol Head Neck Surg,2002.127(3):p.169-76.
67.Katakura,T.,et al.,A combination therapy using IL-12 and soluble IL-4 receptor on herpes simplex virus Type 1 infection in a human-SCID chimera model of thermal injury.Clin Immunol,2002.105(3):p.363-70.
68.Kobayashi,M.,et al.,Effect of a combination therapy between IL-12 and soluble IL-4receptor(sIL-4R) on Candida albicans and herpes simplex virus type Ⅰ infections in thermally injured mice.Can J Microbiol,2002.48(10):p.886-94.
69.Mitsuyasu,H.,et al.,Ile50Val variant of IL4R alpha upregulates IgE synthesis and associates with atopic asthma.Nat Genet,1998.19(2):p.119-20.
70,Thompson,J.D.,et al.,The CLUSTAL_X windows interface:flexible strategies for multiple sequence alignment aided by quality analysis tools.Nucleic Acids Res,1997.25(24):p.4876-82.
71.Saitou,N.and M.Nei,The neighbor-joining method:a new method for reconstructing phylogenetic trees.Mol Biol Evol,1987.4(4):p.406-25.
72.Falquet,L.,et al.,The PROSITE database,its status in 2002.Nucleic Acids Res,2002.30(1):p.235-8.
73.Ho,I.C.,et al.,The proto-oncogene c-maf is responsible for tissue-specific expression of interleukin-4.Cell,1996.85(7):p.973-83.
74.Ho,I.C.and L.H.Glimcher,Transcription:tantalizing times for T cells.Cell,2002.109 Suppl:p.S109-20.
75.Zhang,D.H.,et al.,Transcription factor GATA-3 is differentially expressed in murine Th1 and Th2 cells and controls Th2-specific expression of the interleukin-5 gene.J Biol Chem,1997.272(34):p.21597-603.
76.Zheng,W.and R.A.Flavell,The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells.Cell,1997.89(4):p.587-96.
77.Kim,J.I.,et al.,The transcription factor c-Maf controls the production of interleukin-4 but not other Th2 cytokines.Immunity,1999.10(6):p.745-51.
78.Gilmour,J.and P.Lavender,Control of IL-4 expression in T helper 1 and 2 cells.Immunology,2008.124(4):p.437-44.