LPS诱导的免疫相关基因在免疫系统中潜在功能的研究
发布时间：2018-09-11 20:45
【摘要】：外界病原微生物可以从机体的不同部位侵入,并通过各种机制诱发疾病的产生。当外界致病微生物穿越了人体的上皮屏障,并在宿主的组织内开始大量复制时,会被定居在组织中的单核吞噬细胞以及巨噬细胞所识别。巨噬细胞在机体遭遇病原微生物的入侵时能够迅速做出反应,一方面进行吞噬病原体,另一方面能够释放大量的效应分子引起组织内的炎症反应,进而控制感染。机体的免疫系统对抗原识别过程依赖于不同模式识别受体(pattern recognition receptors, PRR)来完成的,如TLR(sToll like receptors)、NLRs (NOD-like receptors)、RLH(RIG-like helicases)等。Toll样受体作为先天性免疫系统中的重要组成部分,主要通过识别病原微生物表面的病原相关分子模式(pathogen-associated molecular patterns, PAMPs)来启动免疫反应,进而清除外来抗原。 脂多糖LPS(lipopolysaccharide)是革兰氏阴性菌细胞壁的组成成分,能够引起多种免疫细胞发生形态、功能以及胞内基因表达的变化,并导致宿主细胞因子失控性地表达,介导严重感染、器官损伤以及败血症休克等多种疾病的产生。研究表明,LPS能够被细胞表面的TLR4分子所识别,并将信号传递至胞内,激活下游的多条信号,如MAPK(Mitogen activated protein kinase)、NF-κB(Nuclear factor kappa-light-chain-enhancer of activated B cells)、AKT/PKB等信号途径,引发细胞产生一系列的固有免疫分子,如细胞因子(cytokine)和趋化因子(chemokines),包括IL-1(Interleukin-1)、TNF-α(Tumor necrosis factor-α)、IL-6 (Interleukin-6)、IL-12(Interleukin-12)及趋化因子IL-8 (Interleukin-8)等等。TLR家族不仅能够启动天然免疫应答,控制炎症反应的性质、强度以及持续时间,还可以通过上调抗原提呈细胞(antigen-presenting cell,APC)表面的共刺激分子以及MHCⅡ的表达,促进APC的成熟,参与抗原特异性免疫应答,尤其是Th1型反应的产生,调节获得性免疫应答的强度以及类型,成为连接天然免疫(innate immunity)和获得性免疫应答(adaptive immunity)的枢纽。但是,TLR信号过度活化或着活化不足,都会导致机体功能异常以及疾病的发生,因此其调控过程受到其它很多信号通路的正向或负向调控,使之维持适度的活化平衡。 在LPS诱导的TLR4信号转导途径中,多种蛋白参与其调节过程。目前只是对其中一些关键的分子研究的较深入。LPS刺激细胞能够引起众多基因表达量的变化,参与其信号调节过程。如何筛选到这些基因以及探索其功能仍是任重道远。本课题组通过对比小鼠活化与非活化状态的脾细胞EST数据库,筛选到一些在可能在免疫系统中具有潜在功能的基因。并针对其中的部分基因做了一定的研究。本文重点介绍Rab10、Sik1这两个基因。实验通过LPS刺激树突状细胞(dendritic cell,DC),发现随着LPS刺激时间的不同,Rab10及Sik1都呈现一定的变化趋势,暗示Rab10及Sik1在可能在免疫调节中扮演一定的角色。在Rab10基因的研究过程中,实验通过构建了Rab10过表达的质粒在RAW264.7细胞、原代巨噬细胞中瞬时过表达Rab10以及构建了Rab10稳定沉默的细胞系Rab10iA及Rab10KD251,在体外研究Rab10在免疫调节中的功能。最后通过小鼠急性肺损伤模型在体内验证了Rab10在免疫系统中可能的功能。研究结果表明,Rab10蛋白在LPS诱导的TLR4信号转导过程中具有重要的作用。Rab10蛋白能够提高IFN-?、NF-κB启动子的活性,增强MAPK、NF-κB、IRF3信号途径,上调LPS诱导的相关细胞因子如TNF-a、IL-6、IFN-?等的表达,其具体机制是通过辅助TLR4从高尔基体转运至细胞膜,增强LPS诱导的TLR4信号转导途径,从而调节免疫应答。 在Sik1基因的研究过程中实验构建了Sik1过表达以及RNA干扰的腺病毒载体,在RAW264.7中过表达Sik1以及在DC细胞中进行RNA干扰等实验。研究结果表明,SIK1蛋白能够增强LPS诱导的MAPK、NF-κB信号途径,提高NF-κB的转录活性,促进促炎症因子IL-6、IL-12、TNF-a的表达,抑制抗炎因子IL-10的表达。
[Abstract]:External pathogenic microorganisms can invade different parts of the body and induce disease through various mechanisms. When pathogenic microorganisms cross the human epithelial barrier and begin to replicate in large quantities in host tissues, they are recognized by mononuclear phagocytes and macrophages that settle in the tissues. Macrophages are encountered in the body. When invaded by pathogenic microorganisms, the immune system can react quickly, on the one hand, phagocytosis pathogens, on the other hand, release a large number of effector molecules to cause inflammation in tissues, and then control infection. TLR (sToll like receptors), NLRs (NOD-like receptors), RLH (RIG-like helicases) and so on. Toll-like receptors, as an important component of the innate immune system, initiate the immune response mainly by identifying pathogen-associated molecular patterns (PAMPs) on the surface of pathogenic microorganisms, and then clear the immune system. In addition to foreign antigens.
Lipopolysaccharide (LPS) is a component of the cell wall of Gram-negative bacteria. LPS can cause changes in the morphology, function and intracellular gene expression of many immune cells, and lead to uncontrolled expression of host cytokines, mediating the production of severe infections, organ damage and septic shock. S can be recognized by TLR4 molecules on the cell surface and transmitted to the cell, activating downstream signals, such as MAPK (Mitogen activated protein kinase), NF-kappa (nuclear factor kappa-light-chain-enhancer of activated B cells), AKT/PKB and other signaling pathways, triggering the production of a series of intrinsic immune molecules, such as cytokines. Cykines and chemokines, including IL-1 (Interleukin-1), TNF-alpha (Tumor necrosis factor-alpha), IL-6 (Interleukin-6), IL-12 (Interleukin-12) and chemokine IL-8 (Interleukin-8), etc. The TLR family can not only initiate the natural immune response, but also control the nature, intensity and duration of the inflammatory response, and can also be passed through. Upregulate the expression of co-stimulatory molecules and MHC II on the surface of antigen-presenting cell (APC), promote the maturation of APC, participate in antigen-specific immune response, especially the production of Th1 type response, regulate the intensity and type of acquired immune response, and become a link between innate immunity and acquired immune response (a). However, excessive activation or inadequate activation of TLR signals can lead to dysfunction and disease, so its regulation process is regulated by many other signaling pathways in a positive or negative way to maintain a moderate activation balance.
Many proteins are involved in the regulation of TLR4 signal transduction pathway induced by LPS. At present, only some of the key molecules have been studied deeply. LPS-stimulated cells can cause many changes in gene expression and participate in the signal regulation process. By comparing the activated and inactivated EST databases of spleen cells in mice, a number of genes with potential functions in the immune system were screened and some of them were studied. Rab10 and Sik1 showed a tendency to change with LPS stimulation time, suggesting that Rab10 and Sik1 may play a role in immune regulation. In the study of Rab10 gene, Rab10 overexpression plasmid was constructed in RAW264.7 cells, Rab10 overexpression in primary macrophages and Rab10 overexpression in primary macrophages. The stable silent cell lines Rab10iA and Rab10KD251 were used to study the function of Rab10 in immune regulation in vitro. Finally, the possible function of Rab10 in the immune system was verified by acute lung injury model in vivo. The results showed that Rab10 protein played an important role in LPS-induced TLR4 signal transduction. Increase the activity of IFN-, NF-kappa B promoter, enhance MAPK, NF-kappa B, IRF3 signaling pathway, up-regulate the expression of LPS-induced cytokines such as TNF-a, IL-6, IFN-? By assisting TLR4 transport from the Golgi apparatus to the cell membrane and enhancing the LPS-induced TLR4 signal transduction pathway, thereby regulating the immune response.
Sik1 overexpression and RNA interference adenovirus vectors were constructed during the study of Sik1 gene. Overexpression of Sik1 in RAW264.7 and RNA interference in DC cells were tested. The results showed that SIK1 protein could enhance LPS-induced MAPK, NF-kappa B signaling pathway, enhance the transcriptional activity of NF-kappa B, and promote pro-inflammatory factors IL-6, IL-kappa B. 12, the expression of TNF-a inhibits the expression of IL-10.
【学位授予单位】：浙江理工大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392
[Abstract]:External pathogenic microorganisms can invade different parts of the body and induce disease through various mechanisms. When pathogenic microorganisms cross the human epithelial barrier and begin to replicate in large quantities in host tissues, they are recognized by mononuclear phagocytes and macrophages that settle in the tissues. Macrophages are encountered in the body. When invaded by pathogenic microorganisms, the immune system can react quickly, on the one hand, phagocytosis pathogens, on the other hand, release a large number of effector molecules to cause inflammation in tissues, and then control infection. TLR (sToll like receptors), NLRs (NOD-like receptors), RLH (RIG-like helicases) and so on. Toll-like receptors, as an important component of the innate immune system, initiate the immune response mainly by identifying pathogen-associated molecular patterns (PAMPs) on the surface of pathogenic microorganisms, and then clear the immune system. In addition to foreign antigens.
Lipopolysaccharide (LPS) is a component of the cell wall of Gram-negative bacteria. LPS can cause changes in the morphology, function and intracellular gene expression of many immune cells, and lead to uncontrolled expression of host cytokines, mediating the production of severe infections, organ damage and septic shock. S can be recognized by TLR4 molecules on the cell surface and transmitted to the cell, activating downstream signals, such as MAPK (Mitogen activated protein kinase), NF-kappa (nuclear factor kappa-light-chain-enhancer of activated B cells), AKT/PKB and other signaling pathways, triggering the production of a series of intrinsic immune molecules, such as cytokines. Cykines and chemokines, including IL-1 (Interleukin-1), TNF-alpha (Tumor necrosis factor-alpha), IL-6 (Interleukin-6), IL-12 (Interleukin-12) and chemokine IL-8 (Interleukin-8), etc. The TLR family can not only initiate the natural immune response, but also control the nature, intensity and duration of the inflammatory response, and can also be passed through. Upregulate the expression of co-stimulatory molecules and MHC II on the surface of antigen-presenting cell (APC), promote the maturation of APC, participate in antigen-specific immune response, especially the production of Th1 type response, regulate the intensity and type of acquired immune response, and become a link between innate immunity and acquired immune response (a). However, excessive activation or inadequate activation of TLR signals can lead to dysfunction and disease, so its regulation process is regulated by many other signaling pathways in a positive or negative way to maintain a moderate activation balance.
Many proteins are involved in the regulation of TLR4 signal transduction pathway induced by LPS. At present, only some of the key molecules have been studied deeply. LPS-stimulated cells can cause many changes in gene expression and participate in the signal regulation process. By comparing the activated and inactivated EST databases of spleen cells in mice, a number of genes with potential functions in the immune system were screened and some of them were studied. Rab10 and Sik1 showed a tendency to change with LPS stimulation time, suggesting that Rab10 and Sik1 may play a role in immune regulation. In the study of Rab10 gene, Rab10 overexpression plasmid was constructed in RAW264.7 cells, Rab10 overexpression in primary macrophages and Rab10 overexpression in primary macrophages. The stable silent cell lines Rab10iA and Rab10KD251 were used to study the function of Rab10 in immune regulation in vitro. Finally, the possible function of Rab10 in the immune system was verified by acute lung injury model in vivo. The results showed that Rab10 protein played an important role in LPS-induced TLR4 signal transduction. Increase the activity of IFN-, NF-kappa B promoter, enhance MAPK, NF-kappa B, IRF3 signaling pathway, up-regulate the expression of LPS-induced cytokines such as TNF-a, IL-6, IFN-? By assisting TLR4 transport from the Golgi apparatus to the cell membrane and enhancing the LPS-induced TLR4 signal transduction pathway, thereby regulating the immune response.
Sik1 overexpression and RNA interference adenovirus vectors were constructed during the study of Sik1 gene. Overexpression of Sik1 in RAW264.7 and RNA interference in DC cells were tested. The results showed that SIK1 protein could enhance LPS-induced MAPK, NF-kappa B signaling pathway, enhance the transcriptional activity of NF-kappa B, and promote pro-inflammatory factors IL-6, IL-kappa B. 12, the expression of TNF-a inhibits the expression of IL-10.
【学位授予单位】：浙江理工大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392
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1 周晓荣;周芸;周光炎;季玉红;汪晓莺;;SAHA抑制经Ⅱ型胶原体内致敏的Th1细胞增殖与凋亡[J];现代免疫学;2009年06期
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1 陈曙平;曲古抑菌素A对伊马替尼耐药慢性粒细胞白血病细胞的研究[D];中南大学;2012年
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1 张s，
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