LPS誘導(dǎo)的免疫相關(guān)基因在免疫系統(tǒng)中潛在功能的研究
發(fā)布時(shí)間：2018-09-11 20:45
【摘要】：外界病原微生物可以從機(jī)體的不同部位侵入,并通過(guò)各種機(jī)制誘發(fā)疾病的產(chǎn)生。當(dāng)外界致病微生物穿越了人體的上皮屏障,并在宿主的組織內(nèi)開(kāi)始大量復(fù)制時(shí),會(huì)被定居在組織中的單核吞噬細(xì)胞以及巨噬細(xì)胞所識(shí)別。巨噬細(xì)胞在機(jī)體遭遇病原微生物的入侵時(shí)能夠迅速做出反應(yīng),一方面進(jìn)行吞噬病原體,另一方面能夠釋放大量的效應(yīng)分子引起組織內(nèi)的炎癥反應(yīng),進(jìn)而控制感染。機(jī)體的免疫系統(tǒng)對(duì)抗原識(shí)別過(guò)程依賴(lài)于不同模式識(shí)別受體(pattern recognition receptors, PRR)來(lái)完成的,如TLR(sToll like receptors)、NLRs (NOD-like receptors)、RLH(RIG-like helicases)等。Toll樣受體作為先天性免疫系統(tǒng)中的重要組成部分,主要通過(guò)識(shí)別病原微生物表面的病原相關(guān)分子模式(pathogen-associated molecular patterns, PAMPs)來(lái)啟動(dòng)免疫反應(yīng),進(jìn)而清除外來(lái)抗原。 脂多糖LPS(lipopolysaccharide)是革蘭氏陰性菌細(xì)胞壁的組成成分,能夠引起多種免疫細(xì)胞發(fā)生形態(tài)、功能以及胞內(nèi)基因表達(dá)的變化,并導(dǎo)致宿主細(xì)胞因子失控性地表達(dá),介導(dǎo)嚴(yán)重感染、器官損傷以及敗血癥休克等多種疾病的產(chǎn)生。研究表明,LPS能夠被細(xì)胞表面的TLR4分子所識(shí)別,并將信號(hào)傳遞至胞內(nèi),激活下游的多條信號(hào),如MAPK(Mitogen activated protein kinase)、NF-κB(Nuclear factor kappa-light-chain-enhancer of activated B cells)、AKT/PKB等信號(hào)途徑,引發(fā)細(xì)胞產(chǎn)生一系列的固有免疫分子,如細(xì)胞因子(cytokine)和趨化因子(chemokines),包括IL-1(Interleukin-1)、TNF-α(Tumor necrosis factor-α)、IL-6 (Interleukin-6)、IL-12(Interleukin-12)及趨化因子IL-8 (Interleukin-8)等等。TLR家族不僅能夠啟動(dòng)天然免疫應(yīng)答,控制炎癥反應(yīng)的性質(zhì)、強(qiáng)度以及持續(xù)時(shí)間,還可以通過(guò)上調(diào)抗原提呈細(xì)胞(antigen-presenting cell,APC)表面的共刺激分子以及MHCⅡ的表達(dá),促進(jìn)APC的成熟,參與抗原特異性免疫應(yīng)答,尤其是Th1型反應(yīng)的產(chǎn)生,調(diào)節(jié)獲得性免疫應(yīng)答的強(qiáng)度以及類(lèi)型,成為連接天然免疫(innate immunity)和獲得性免疫應(yīng)答(adaptive immunity)的樞紐。但是,TLR信號(hào)過(guò)度活化或著活化不足,都會(huì)導(dǎo)致機(jī)體功能異常以及疾病的發(fā)生,因此其調(diào)控過(guò)程受到其它很多信號(hào)通路的正向或負(fù)向調(diào)控,使之維持適度的活化平衡。 在LPS誘導(dǎo)的TLR4信號(hào)轉(zhuǎn)導(dǎo)途徑中,多種蛋白參與其調(diào)節(jié)過(guò)程。目前只是對(duì)其中一些關(guān)鍵的分子研究的較深入。LPS刺激細(xì)胞能夠引起眾多基因表達(dá)量的變化,參與其信號(hào)調(diào)節(jié)過(guò)程。如何篩選到這些基因以及探索其功能仍是任重道遠(yuǎn)。本課題組通過(guò)對(duì)比小鼠活化與非活化狀態(tài)的脾細(xì)胞EST數(shù)據(jù)庫(kù),篩選到一些在可能在免疫系統(tǒng)中具有潛在功能的基因。并針對(duì)其中的部分基因做了一定的研究。本文重點(diǎn)介紹Rab10、Sik1這兩個(gè)基因。實(shí)驗(yàn)通過(guò)LPS刺激樹(shù)突狀細(xì)胞(dendritic cell,DC),發(fā)現(xiàn)隨著LPS刺激時(shí)間的不同,Rab10及Sik1都呈現(xiàn)一定的變化趨勢(shì),暗示Rab10及Sik1在可能在免疫調(diào)節(jié)中扮演一定的角色。在Rab10基因的研究過(guò)程中,實(shí)驗(yàn)通過(guò)構(gòu)建了Rab10過(guò)表達(dá)的質(zhì)粒在RAW264.7細(xì)胞、原代巨噬細(xì)胞中瞬時(shí)過(guò)表達(dá)Rab10以及構(gòu)建了Rab10穩(wěn)定沉默的細(xì)胞系Rab10iA及Rab10KD251,在體外研究Rab10在免疫調(diào)節(jié)中的功能。最后通過(guò)小鼠急性肺損傷模型在體內(nèi)驗(yàn)證了Rab10在免疫系統(tǒng)中可能的功能。研究結(jié)果表明,Rab10蛋白在LPS誘導(dǎo)的TLR4信號(hào)轉(zhuǎn)導(dǎo)過(guò)程中具有重要的作用。Rab10蛋白能夠提高IFN-?、NF-κB啟動(dòng)子的活性,增強(qiáng)MAPK、NF-κB、IRF3信號(hào)途徑,上調(diào)LPS誘導(dǎo)的相關(guān)細(xì)胞因子如TNF-a、IL-6、IFN-?等的表達(dá),其具體機(jī)制是通過(guò)輔助TLR4從高爾基體轉(zhuǎn)運(yùn)至細(xì)胞膜,增強(qiáng)LPS誘導(dǎo)的TLR4信號(hào)轉(zhuǎn)導(dǎo)途徑,從而調(diào)節(jié)免疫應(yīng)答。 在Sik1基因的研究過(guò)程中實(shí)驗(yàn)構(gòu)建了Sik1過(guò)表達(dá)以及RNA干擾的腺病毒載體,在RAW264.7中過(guò)表達(dá)Sik1以及在DC細(xì)胞中進(jìn)行RNA干擾等實(shí)驗(yàn)。研究結(jié)果表明,SIK1蛋白能夠增強(qiáng)LPS誘導(dǎo)的MAPK、NF-κB信號(hào)途徑,提高NF-κB的轉(zhuǎn)錄活性,促進(jìn)促炎癥因子IL-6、IL-12、TNF-a的表達(dá),抑制抗炎因子IL-10的表達(dá)。
[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.
【學(xué)位授予單位】：浙江理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：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.
【學(xué)位授予單位】：浙江理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R392
【共引文獻(xiàn)】
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1 張s，
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