SHP-1及热休克调节单核巨噬细胞Toll样受体信号转导研究

发布时间：2018-03-01 03:13

本文关键词： Toll样受体单核巨噬细胞 SHP-1 热休克 CpG ODN　出处：《浙江大学》2005年博士论文　论文类型：学位论文

【摘要】：Toll样受体(Toll like receptors,TLRs)作为一种重要的模式识别受体,主要表达于巨噬细胞和树突状细胞表面。TLRs是Ⅰ型跨膜蛋白,属于IL-1R/TLR受体超家族,通过辨认识别生物体内保守的病原相关分子模式(Pathogen-associated molecule patterns,PAMPs),活化巨噬细胞和树突状细胞,产生细胞因子和趋化因子,启动天然免疫应答,构成机体免疫反应的第一道防线。Toll蛋白最早发现在调控果蝇的胚胎发育过程中起重要作用,目前,在哺乳动物细胞中相继发现并克隆了11种TLR分子,选择性识别病原体中特定的分子结构,以抵抗病原菌的入侵,TLR家族成员的缺失导致机体对病原体高度易感。本课题研究了单核巨噬细胞酪氨酸磷酸酶和热休克对Toll样受体信号转导的调节以及相应的作用机制。第一部分 SHP-1参与巨噬细胞TLR9信号转导通路的研究细菌DNA不仅是遗传的物质基础,其中某些特定序列还具有免疫活性。人工合成CpG寡核苷酸(CpG-Oligodeoxynucleotides,CpG ODN)能模拟细菌DNA的免疫活性效应。CpG ODN可活化自然杀伤细胞(Natural killer cell,NK细胞)、单核/巨噬细胞、树突状细胞、T细胞等,活化后的细胞可分泌IL-1、IL-6、TNFa等细胞因子、趋化因子、NO等免疫效应分子,诱导天然免疫应答。CpG ODN的辨认和信号传导通过TLR9所介导。TLR9识别配体后,通过MyD88-IRAK-TRAF6激酶途径募集接头蛋白髓系分化蛋白88(MyD88),结合并活化IL-1受体相关激酶(IRAKs)、肿瘤坏死因子受体相关因子6(TRAF6),活化下游MAPKs和NF-κB诱导激酶(NIK)、IKK、IκBa,活化后的NF-κB入核引起特定基因的表达。信号转导过程中许多信号分子相互作用,使信号转导保持一定的强度能顺利传导并能及时终止。SHP-1酪氨酸磷酸酶SHP-1(Src homology 2 domain containingphosphatase 1)最早也命名为SHPTP-1,SHP,HCP和PTP1C,是个胞内酪氨酸磷酸酶,主要表达于造血细胞。SHP-1含两个N端SH2区,单个磷酸酶区,以及C端尾部的两个酪氨酸磷酸化区,是个淋巴细胞、巨噬细胞内常见的信号调节分子。本实验浙江大学博士学位论文研究了在CpG ODN活化巨噬细胞TLRg诱导产生细胞因子等细胞免疫反应过程中, SHP一1参与的调节作用及其可能的作用机制。我们构建了SHP一1高表达载体,PCR定点突变法构建SHP一1催化活性突变载体 (C4535),经测序鉴定正确。合成SHP一1 RNA干扰片段,转染RAW264.7细胞以抑制 SHP一1蛋白合成。G418筛选稳定表达SHP一1和催化活性突变mu一SHP一1 RAW细胞, 经CpG ODN刺激后,发现稳定高表达SHP一1的巨噬细胞的细胞因子IL6的分泌显著增加,而对于SHP一1催化活性突变的巨噬细胞,经CpG ODN刺激后其IL一6的分泌水平依然升高,与高表达SHP一1巨噬细胞相比没有明显变化。而RNA干扰SHP一1蛋白表达后,IL6的分泌水平下降。荧光素酶报告基因检测显示SHP一1高表达的巨噬细胞经CpG ODN刺激后NF一忍的活化水平增高。这些实验现象表明SHP一1高表达可能促进CpG ODN刺激后NF一沼的活化,从而促进细胞因子IL一6的分泌。而催化活性的高低与SHP一1促进细胞因子的表达关系不大。为进一步探讨SHP一1促进CpG ODN刺激巨噬后IL一6分泌的原因,检测了MApK活化水平的改变。CpG ODN刺激后,发现SHP一1 高表达的巨噬细胞ERK、JNK的磷酸化水平没有改变,而p38的磷酸化水平显著增高, 说明sHP一1促进IL6的分泌可能部分与p38的磷酸化水平增高有关。免疫共沉淀进一步检测了cpG ODN刺激后与SHP一1在巨噬细胞内相互作用的一些信号分子,发现 IKK、TRA王6与SHP一1形成蛋白复合物,显示SHP一1可能通过与IKK、TRAF6结合活化NF一虑,从而促进IL6的分泌。上述实验结果表明,SHP一1促进CpG ODN刺激巨噬细胞IL6的表达,其催化酶活性的下降对IL6的表达没有明显影响,而RNA干扰后IL6的分泌水平下降,显示 SHP一1的表达参与CpG ODN刺激后巨噬细胞p38、NF一虑的活化,而使IL一6的分泌增高,表明SHP一1是个参与CPG ODN江LRg信号通路一个重要的胞内调控分子。本实验结果为深入认识cpG oDN月1,R9介导的信号转导的调控提供了一个新的机制。第二部分热休克调节单核细胞Toll样受体信号转导研究热休克能通过诱导热休克蛋白(Heat shock protein:3,HSPs)等应激蛋白诱导机体的固有免疫应答。免疫反应中抗原递呈细胞(Antigen一Presenting een,APC)通过调节病原识别受体(pathogen reeo邵ition reeeptors,pRRs),识别病原相关的分子模式(RAMPs)
[Abstract]:Toll like receptors (Toll like receptors, TLRs) as a kind of important pattern recognition receptors expressed mainly in macrophages and dendritic cell surface.TLRs is a type I transmembrane protein, belongs to the IL-1R/TLR superfamily, by identifying the pathogenic organisms related to conserved recognition molecules (Pathogen-associated molecule patterns, PAMPs model), and activated macrophages dendritic cells, cytokines and chemokines, initiate innate immune response, the immune response constitutes the first line of defense.Toll protein was found to play an important role in the process, regulation of Drosophila embryonic development at present, have been found and cloned 11 TLR molecules in mammalian cells. The specific molecular structure of selective recognition of pathogens, to resist the invasion of pathogens, the lack of a member of the TLR family will cause the body to be highly susceptible to pathogens. The subject of mononuclear macrophage The regulation of phagocytic tyrosine phosphatase and heat shock to the signal transduction of Toll like receptor and the corresponding mechanism of action.
Part one SHP-1 participates in the study of TLR9 signal transduction pathway in macrophages
DNA is not only based on the genetic material of bacteria, which also has a certain sequence of immune activity of synthetic CpG oligonucleotides (CpG-Oligodeoxynucleotides, CpG ODN) to.CpG ODN immune effect simulation of bacterial DNA can activate natural killer cells (Natural killer cell, NK cells), monocytes / macrophages, dendritic cells, T cells and so on. The activated cells can secrete IL-1, IL-6, TNFa and other cytokines, chemokines, NO and other immune effector molecules induced by the innate immune response.CpG ODN recognition and signal transduction mediated by TLR9.TLR9 ligand recognition, through the MyD88-IRAK-TRAF6 adaptor protein kinase pathway to raise myeloid differentiation protein 88 (MyD88), combined with and the activation of IL-1 receptor associated kinase (IRAKs), tumor necrosis factor receptor associated factor 6 (TRAF6), activation of downstream MAPKs and NF- K B induced kinase (NIK), IKK, I K Ba activation of NF- kappa B nuclear special cause The expression of the fixed gene.
The interaction of many signal molecules in signal transduction, signal transduction to maintain a certain strength of the smooth transmission and timely termination of.SHP-1 tyrosine phosphatase SHP-1 (Src homology 2 domain containingphosphatase 1) was also named as SHPTP-1, SHP, HCP and PTP1C, is an intracellular tyrosine phosphatase, mainly expressed in hematopoietic cells containing.SHP-1 two N SH2, a single phosphatase domain two tyrosine phosphorylation and C tail, is a common signal molecular regulation of lymphocytes, macrophages in vitro.
Doctoral Dissertation of Zhejiang University
The immunoreaction of cytokines, such as cytokines, induced by CpG ODN activated macrophage TLRg, was studied.
The regulatory role of SHP 1 participation and its possible mechanism of action.
We constructed a SHP 1 high expression vector and PCR fixed-point mutation method for the construction of SHP 1 catalytic active mutation carrier
(C4535), correct by sequencing, synthesis of SHP 1 RNA interference fragments, transfection of RAW264.7 cells to inhibit
SHP 1 protein synthesis.G418 screening stable expression of SHP 1 1 and catalytic activity mutation mu SHP 1 RAW cells,
After CpG ODN stimulation, it was found that the secretion of cytokine IL6, which was stable and high expression of SHP 1 macrophages, was significant
Increase, and for macrophages with SHP 1 catalytic activity mutation, the IL 6 secreted water after the stimulation of CpG ODN
The level still increased, and there was no significant change compared with the high expression of SHP 1 1 macrophages. RNA interfered with SHP 1 protein.
After expression, the secretory level of IL6 decreased. The luciferase reporter gene detection showed the high expression of SHP 1 macrophages
The activation level of NF was increased after CpG ODN stimulation. These experimental phenomena suggest that the high expression of SHP 1 may be promoted.
The activation of NF marsh after the stimulation of CpG ODN stimulates the secretion of cytokine IL 1 6, and the catalytic activity is high
The relationship between low and SHP 1 promoting the expression of cytokines is not significant. To further explore the promotion of SHP 1 1 to promote CpG ODN stimulation
The cause of IL 6 secretion after Mega phagocytosis, detection of MApK activation level changes.CpG ODN stimulation, found SHP 1
The high expression of macrophage ERK, the level of phosphorylation of JNK did not change, and the level of phosphorylation of p38 was significantly higher.
This shows that the possible part of sHP 1 to promote the secretion of IL6 is related to the increased phosphorylation level of p38.
One step detected some of the signal molecules interacting with SHP 1 in macrophages after cpG ODN stimulation.
IKK, TRA King 6 and SHP - 1 forming protein complex, showing that SHP 1 1 may be combined with IKK, TRAF6
The consideration of NF, thus promoting the secretion of IL6.
The results of these experiments showed that SHP 1 promoted CpG ODN to stimulate the expression of IL6 in macrophage, and its catalytic enzyme
The decrease of activity had no significant effect on the expression of IL6, but the secretion level of IL6 decreased after RNA interference.
The expression of SHP 1 participates in the activation of p38, NF in macrophages after CpG ODN stimulation, and increases the secretion of IL 6.
High level, indicating that SHP 1 is an important intracellular regulator involved in the CPG ODN LRg signaling pathway.
The results provide a new mechanism for the in-depth understanding of cpG oDN 1, and the regulation of signal transduction mediated by R9.
Study on the signal transduction of Toll like receptor in the second part of heat shock regulating monocyte
Heat shock can induce the body by inducing stress proteins such as Heat shock protein:3 (HSPs) and other stress proteins
The intrinsic immune response. The antigen presenting cell (Antigen Presenting een, APC) in the immune response is mediated by the regulation of the disease
The original recognition receptor (pathogen reeo Shao ition reeeptors, pRRs), identifying the molecular pattern associated with the pathogen (RAMPs)

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R392.1

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