本文選題：iOPN + Ⅰ型干擾素��； 參考：《山東大學(xué)》2016年博士論文

【摘要】：固有免疫系統(tǒng)作為機(jī)體抵御外界病原體入侵的首道防線,不僅可以激活適應(yīng)性免疫系統(tǒng),同時(shí)也可以直接對(duì)入侵的病原體產(chǎn)生強(qiáng)烈的免疫應(yīng)答,從而殺傷病原體。在病毒入侵時(shí),固有免疫系統(tǒng)可以激活產(chǎn)生多種細(xì)胞因子,其中最主要的是I型干擾素(IFNα/β)。固有免疫系統(tǒng)中的多種的模式識(shí)別受體(PRRs)都與Ⅰ型干擾素的產(chǎn)生相關(guān),包括諸如Toll樣受體(TLRs), RIG-I樣受體(RLRs)和內(nèi)源性DNA感受器等。產(chǎn)生的Ⅰ型干擾素可以進(jìn)而與其受體結(jié)合,活化JAK (Janus kinase)和STAT (Signaltransducers and activators of transcription)信號(hào)通路,進(jìn)而誘導(dǎo)IFN-stimulated genes (ISGs)的表達(dá),最終清除病毒。骨橋蛋白(osteopontin, OPN)又稱為早期T淋巴細(xì)胞活化因子1(early T lymphocyte activation 1, Etal),是一種分泌性的多功能糖蛋白。OPN可以調(diào)節(jié)多種生物體進(jìn)程,包括細(xì)胞分化、粘附、骨重構(gòu)、惡性腫瘤和免疫反應(yīng)。長(zhǎng)期以來,OPN被認(rèn)為是一個(gè)與炎癥過程有關(guān)的潛在的促炎癥細(xì)胞因子,具有促進(jìn)巨噬細(xì)胞分泌IFN-γ和IL-12的作用。近年來,隨著胞內(nèi)形式OPN (iOPN)的發(fā)現(xiàn),OPN在不同免疫細(xì)胞以及免疫反應(yīng)的不同階段中的不同調(diào)控作用逐漸引起人們的重視。而iOPN在固有免疫系統(tǒng)當(dāng)中尤其是抗病毒免疫當(dāng)中的作用還未明確。研究目的：探討OPN,尤其是iOPN是否可以調(diào)節(jié)病毒誘導(dǎo)的Ⅰ型干擾素產(chǎn)生。如若可以調(diào)節(jié)Ⅰ型干擾素產(chǎn)生,闡述其潛在機(jī)制。研究方法：1.首先檢測(cè)不同病毒及刺激劑對(duì)OPN表達(dá)的影響。再利用OPN缺陷型(Spp1-/-)小鼠與野生型(WT)小鼠的腹腔巨噬細(xì)胞,經(jīng)病毒刺激后,檢測(cè)IFNβ以及下游多種代表性ISGs的mRNA水平變化、蛋白分泌水平變化等。通過構(gòu)建全長(zhǎng)型OPN (full length OPN)及胞內(nèi)形式OPN(iOPN)表達(dá)質(zhì)粒并轉(zhuǎn)染進(jìn)HEK293細(xì)胞,利用雙熒光素酶報(bào)告基因,檢測(cè)兩種不同形式OPN對(duì)于調(diào)節(jié)RNA病毒SeV所活化的IFN表達(dá)的影響,并檢測(cè)iOPN質(zhì)粒對(duì)于各種接頭分子所活化的IFN啟動(dòng)子區(qū)活性的調(diào)節(jié)作用。2.利用OPN缺陷型(Sppl-/-)小鼠與野生型(WT)小鼠,通過腹腔注射VSV病毒,檢測(cè)VSV病毒在小鼠臟器中的滴度與含量,反應(yīng)兩類小鼠對(duì)于VSV病毒的抵抗能力的區(qū)別。3.通過利用OPN缺陷型(Spp1-/-)小鼠與野生型(WT)小鼠腹腔巨噬細(xì)胞,以及iOPN過表達(dá)質(zhì)粒轉(zhuǎn)染HEK293細(xì)胞,檢測(cè)經(jīng)SeV刺激后,重要的IFN轉(zhuǎn)錄因子IRF3的活化水平變化。4.經(jīng)實(shí)時(shí)熒光定量PCR (q-PCR)以及雙熒光素酶報(bào)告基因?qū)嶒?yàn),檢測(cè)iOPN質(zhì)粒的靶點(diǎn)分子,并經(jīng)免疫共沉淀(co-IP)以及免疫熒光等實(shí)驗(yàn)檢測(cè)iOPN與靶點(diǎn)分子的結(jié)合情況。5.明確靶點(diǎn)分子后,利用不同類型的泛素分子質(zhì)粒,共轉(zhuǎn)染iOPN及靶點(diǎn)分子進(jìn)HEK293細(xì)胞,檢測(cè)其是否發(fā)生泛素化水平的變化,以及靶點(diǎn)分子的穩(wěn)定性是否發(fā)生變化。6.靶點(diǎn)分子若發(fā)生K48位泛素化水平的變化以及其穩(wěn)定性若發(fā)生變化,說明iOPN可能可以調(diào)節(jié)靶點(diǎn)分子的穩(wěn)定性。通過對(duì)靶點(diǎn)已知的泛素化酶以及去泛素化酶進(jìn)行篩選,尋找可能的作用機(jī)制,并通過細(xì)胞水平以及體外翻譯系統(tǒng)對(duì)結(jié)論進(jìn)行實(shí)驗(yàn)性確認(rèn)。7.通過利用細(xì)胞過表達(dá)載體以及慢病毒表達(dá)載體構(gòu)建iOPN-WT、iOPN-N、iOPN-C重組過表達(dá)載體,通過體外翻譯系統(tǒng)實(shí)驗(yàn)或?qū)PN缺陷型小鼠腹腔巨噬細(xì)胞進(jìn)行iOPN回補(bǔ)實(shí)驗(yàn),明確iOPN具體發(fā)生功能的區(qū)域。研究結(jié)果：1.首先,在利用RNA病毒VSV(水泡性口炎病毒)、SeV(仙臺(tái)病毒)以及DNA病毒HSV(單純皰疹病毒)等病毒刺激野生型小鼠腹腔巨噬細(xì)胞不同時(shí)間點(diǎn)后,發(fā)現(xiàn)OPN的表達(dá)都有逐漸升高的趨勢(shì)；其次,在OPN缺陷型小鼠以及野生型小鼠巨噬細(xì)胞中加入SeV或VSV刺激不同時(shí)間后,發(fā)現(xiàn)相比較于野生型組,OPN缺陷型小鼠腹腔巨噬細(xì)胞產(chǎn)生IFNβ的能力都明顯降低；將構(gòu)建成功的全長(zhǎng)型OPN表達(dá)載體與iOPN表達(dá)載體分別轉(zhuǎn)染進(jìn)HEK293后經(jīng)SeV刺激后,發(fā)現(xiàn)二種質(zhì)粒都可以使IFNβ表達(dá)增強(qiáng),但是iOPN表達(dá)載體組明顯強(qiáng)于全長(zhǎng)型OPN表達(dá)載體組,并且利用OPN封閉抗體封閉分泌型OPN后,IFNβ的產(chǎn)生無(wú)明顯變化,說明全長(zhǎng)型OPN所表達(dá)的分泌性O(shè)PN可能并不參與調(diào)節(jié)IFNβ的增強(qiáng)過程；iOPN也可以明顯增強(qiáng)RIG-I、MDA5、TRIF以及STINGcGAS等接頭分子所介導(dǎo)的IFNβ啟動(dòng)子區(qū)的活化。2.OPN缺陷型小鼠相比較于野生型小鼠,會(huì)產(chǎn)生較少的IFNβ,進(jìn)而造成此組巨噬細(xì)胞或成年小鼠在經(jīng)VSV病毒感染后,VSV病毒的滴度以及VSV mRNA水平以及VSV病毒的蛋白表達(dá)程度更高。并且OPN缺陷型小鼠組在大劑量VSV病毒感染后,生存率明顯低于野生型小鼠組。3.前述實(shí)驗(yàn)結(jié)果證明OPN,尤其是iOPN的確可以正調(diào)IFNβ的產(chǎn)生,又由于IRF3是重要的調(diào)控IFNβ表達(dá)的轉(zhuǎn)錄因子,我們進(jìn)而研究IRF3的活化水平是否有變化。我們發(fā)現(xiàn)無(wú)論是在啟動(dòng)子區(qū)結(jié)合、磷酸化、二聚化以及入核等方面,iOPN都可以起到正調(diào)作用,說明iOPN的確是通過影響IRF3轉(zhuǎn)錄因子的活化進(jìn)而增強(qiáng)了IFNβ的表達(dá)。4.為了明確iOPN作用的靶點(diǎn),我們經(jīng)報(bào)告基因以及realtime-PCR等技術(shù)發(fā)現(xiàn)iOPN作用的靶點(diǎn)可能存在于MAVS以及TBKl附近。通過免疫共沉淀我們發(fā)現(xiàn)iOPN可以特異性與TRAF3結(jié)合,并且這一結(jié)論也經(jīng)免疫熒光以及體外蛋白結(jié)合實(shí)驗(yàn)驗(yàn)證。5.在知道作用靶點(diǎn)為TRAF3后,我們進(jìn)一步研究可能的機(jī)制。泛素化調(diào)節(jié)作用在機(jī)體中具有舉足輕重的作用,我們首先研究下iOPN是否會(huì)影響TRAF3的泛素化。結(jié)果發(fā)現(xiàn),iOPN可以抑制TRAF3發(fā)生K48位泛素化修飾,即iOPN可以起到穩(wěn)定TRAF3表達(dá)的作用。無(wú)論是在HEK293細(xì)胞中過表達(dá)iOPN,或是在OPN缺陷型的小鼠腹腔巨噬細(xì)胞,iOPN的過表達(dá)或缺失都會(huì)導(dǎo)致放線菌酮(CHX)介導(dǎo)的TRAF3的穩(wěn)定性發(fā)生明顯變化。6.由于我們發(fā)現(xiàn)iOPN可以抑制TRAF3發(fā)生K48位泛素化修飾,但由于iOPN本身并沒有去泛素化酶功能,這就提示我們可能iOPN可以抑制某種可介導(dǎo)TRAF3的K48位泛素化作用的E3連接酶與其結(jié)合,或者募集某種可以去除TRAF3的K48位泛素化鏈的去泛素化酶與TRAF3結(jié)合,結(jié)果我們發(fā)現(xiàn)之前有報(bào)道過的TRAF3的去泛素化酶USP25并沒有因?yàn)閕OPN的加入而更加明顯的除去TRAF3上的泛素化鏈,而Triad3A對(duì)于TRAF3的K48位泛素化修飾作用則由于iOPN的加入而明顯受到抑制。進(jìn)而我們發(fā)現(xiàn)iOPN可以明顯的抑制TRAF3與Triad3A的結(jié)合,并且TRAF3的Y440、Q442位點(diǎn)如若被突變則TRAF3不與Triad3A結(jié)合,同時(shí)也不與iOPN結(jié)合。綜上說明iOPN可能是通過結(jié)合了TRAF3進(jìn)而抑制了Triad3A與TRAF3的結(jié)合,iOPN與Triad3A肯能是競(jìng)爭(zhēng)結(jié)合關(guān)系。7.通過構(gòu)建的iOPN-WT以及iOPN-N端或iOPN-C端截短體,我們發(fā)現(xiàn)iOPN的C端可以與TRAF3發(fā)生結(jié)合,并且通過在OPN缺陷型小鼠腹腔巨噬細(xì)胞中進(jìn)行iOPN回補(bǔ)實(shí)驗(yàn),我們進(jìn)一步確定iOPN的C端即足以發(fā)揮結(jié)合TRAF3、抑制TRAF3的K48位泛素化以及增強(qiáng)IFNβ產(chǎn)生的作用�？偨Y(jié)：1.OPN的表達(dá)可被病毒所誘導(dǎo),并且OPN尤其是iOPN可以正調(diào)I型干擾素的產(chǎn)生。2.OPN在體內(nèi)也是Ⅰ型干擾素產(chǎn)生的強(qiáng)力正調(diào)者,可以誘導(dǎo)很強(qiáng)的抗病毒免疫應(yīng)答。3. iOPN可以正調(diào)IRF3轉(zhuǎn)錄因子的活化。4. iOPN可以與重要接頭分子TRAF3特異性結(jié)合。5. IOPN可以調(diào)節(jié)TRAF3的K48位偶聯(lián)的泛素化修飾并且使TRAF3更穩(wěn)定。6. iOPN與Triad3A競(jìng)爭(zhēng)性結(jié)合TRAF3,進(jìn)而影響Triad3A介導(dǎo)的,TRAF3的K48位偶聯(lián)的泛素化。7. iOPN通過C端與TRAF3結(jié)合,并且其C端即可影響TRAF3的泛素化即穩(wěn)定性。創(chuàng)新點(diǎn)及意義：1.本研究首次明確完善地證明iOPN可以參與調(diào)控抗病毒天然免疫反應(yīng),可以正向調(diào)節(jié)IFNβ的產(chǎn)生以及具有明顯的抗病毒功能。這一功能的機(jī)制是由于iOPN通過抑制Triad3A與TRAF3的結(jié)合,從而抑制了Triad3A對(duì)TRAF3的K48位泛素化修飾作用,進(jìn)而導(dǎo)致TRAF3的穩(wěn)定性增強(qiáng),IFNβ的活化進(jìn)而增強(qiáng)。2.本研究提出iOPN可以抑制某分子的泛素化作用這一機(jī)制在國(guó)際上尚屬少見,極大豐富了我們對(duì)于OPN功能的理解。并且我們首次發(fā)現(xiàn)iOPN的C端可以發(fā)揮幾乎全部的抗病毒功能。3.本研究為正向調(diào)控抗病毒免疫信號(hào)通路提供了新的實(shí)驗(yàn)證據(jù),為抗病毒研究以及新藥物靶點(diǎn)的研究提供了新的思路。
[Abstract]:As the first line of defense against the invasion of external pathogens, the innate immune system can not only activate the adaptive immune system, but also produce a strong immune response to the invading pathogen, thus killing the pathogen. In the case of virus invasion, the inherent immune system can activate a variety of cytokines, the most important of which are the most important It is type I interferon (IFN alpha / beta). A variety of pattern recognition receptors (PRRs) in the inherent immune system are associated with the production of type I interferon, including such as Toll like receptor (TLRs), RIG-I like receptor (RLRs) and endogenous DNA receptor. Nsducers and activators of transcription) signaling pathway, which then induces the expression of IFN-stimulated genes (ISGs), and eventually clears the virus. The osteopontin (osteopontin, OPN) is also called the early T lymphocyte activation factor 1 (early), a secretory multifunctional glycoprotein that can regulate a variety of raw materials. Process, including cell differentiation, adhesion, bone remodeling, malignant tumor and immune response. OPN has long been thought to be a potential pro-inflammatory cytokine associated with inflammatory processes, which promotes the secretion of IFN- gamma and IL-12 by macrophages. In recent years, with the discovery of intracellular form OPN (iOPN), OPN is in different immune cells and in different immune cells. The different regulatory roles in the different stages of the immune response have gradually aroused people's attention. And the role of iOPN in the inherent immune system, especially in antiviral immunity, is not clear. The purpose of this study is to explore whether OPN, especially iOPN, can regulate the production of virus induced interferon I. The potential mechanisms are discussed. 1. first, the effects of different viruses and stimulants on the expression of OPN were detected. Then the peritoneal macrophages of OPN deficient (Spp1-/-) mice and wild type (WT) mice were used to detect the changes in the mRNA level of IFN beta and a variety of representative ISGs, and the changes of protein secretion level. The full length OPN (full length OPN) and intracellular form OPN (iOPN) expressed plasmid and transfected into HEK293 cells, using the double luciferase reporter gene to detect the effect of two different forms of OPN on the regulation of IFN expression of the activation of RNA virus SeV, and to detect the regulation of the iOPN plasmids on the activation of the promoter region activated by various connector molecules. 2. using OPN deficient (Sppl-/-) mice and wild type (WT) mice, the titer and content of VSV virus in the mouse organs were detected by intraperitoneal injection of VSV virus. The difference of the resistance ability of the two types of mice to the resistance of VSV virus.3. was expressed by using OPN deficient (Spp1-/-) mice and wild type (WT) mice peritoneal macrophages, and iOPN overexpression. HEK293 cells were transfected by plasmid, and the activation level of the important IFN transcription factor IRF3 was detected by SeV stimulation..4. was detected by real-time fluorescent quantitative PCR (q-PCR) and double luciferase reporter gene test, and the target molecules of iOPN plasmids were detected, and the binding of iOPN and target molecules was detected by immunoprecipitation (co-IP) and immunofluorescence. 5. after identifying the target molecules, using different types of ubiquitin plasmids, CO transfection of iOPN and target molecules into HEK293 cells to detect the changes in the level of ubiquitination, and whether the stability of the target molecules changes if the.6. target molecule changes the level of K48 ubiquitination and its stability changes, indicating iOP N may be able to regulate the stability of the target molecules. Through the screening of ubiquitin and ubiquitination enzymes that are known to the target, the possible mechanisms are found, and the results are experimentally confirmed by the cell level and in vitro translation system, and.7. is constructed by using cell overexpressed carrier and lentivirus expression vector to construct iOPN-WT, iOPN- N, iOPN-C recombinant overexpression vector, through the in vitro translation system experiment or the OPN deficient mouse peritoneal macrophages to carry out the iOPN recharge experiment to clarify the specific region of iOPN function. 1. first, in the use of RNA virus VSV (vesicular stomatitis virus), SeV (Sendai virus) and DNA virus HSV (herpes simplex virus) and other virus spines. After different time points of peritoneal macrophages in wild type mice, it was found that the expression of OPN had a tendency to increase gradually. Secondly, after adding SeV or VSV to the macrophages of OPN deficient mice and wild type mice for different time, it was found that the ability to produce IFN beta in the peritoneal macrophages of OPN deficient mice was better than that in the wild type. The whole long OPN expression vector and iOPN expression vector were transfected into HEK293 respectively. After SeV stimulation, it was found that all two plasmids could enhance the expression of IFN beta, but the iOPN expression vector group was obviously stronger than the full length OPN expression vector group, and the production of IFN beta was not obvious after the OPN closed antibody closed the secretory OPN. It shows that the secretory OPN expressed by the full-length OPN may not be involved in the regulation of the enhancement of IFN beta; iOPN can also significantly enhance the activation of the.2.OPN deficient mice in the IFN beta promoter region mediated by RIG-I, MDA5, TRIF, and STINGcGAS. After VSV virus infection, the titer of the VSV virus and the level of VSV mRNA and the protein expression of the VSV virus are higher in the phagocytic or adult mice. The survival rate of the OPN deficient mice group in the large dose VSV virus infection is significantly lower than that of the wild type mouse group.3. before the experimental results of.3., especially that iOPN can actually be adjusted IFN beta. Because IRF3 is an important transcription factor that regulates the expression of IFN beta, we further investigate whether there is a change in the activation level of IRF3. We found that iOPN can play a positive role in the activation of the promoter region binding, phosphorylation, dimerization, and nucleation, indicating that iOPN does increase by the activation of the IRF3 transcription factor. The expression of IFN beta (.4.) is stronger to identify the target of iOPN action. The target of our reporter gene and realtime-PCR technology may be found near MAVS and TBKl. By immunoprecipitation, we found that iOPN can be specifically associated with TRAF3, and this conclusion is also tested by immunofluorescence and in vitro protein binding experiments. We further study the possible mechanism of.5. after we know that the target target is TRAF3. The regulation of ubiquitination plays an important role in the body. We first study whether iOPN will affect the ubiquitination of TRAF3. It is found that iOPN can inhibit the K48 ubiquitination of TRAF3, that is, iOPN can stabilize TRAF3 expression. Effect. Either over expression of iOPN in HEK293 cells, or in OPN deficient mice peritoneal macrophages, the overexpression or deletion of iOPN causes significant changes in the stability of TRAF3 mediated by actinomycone (CHX),.6. because we found iOPN can inhibit the K48 bit ubiquitination of TRAF3, but because iOPN itself is not ubiquitous The function of the protease suggests that iOPN may inhibit the binding of a E3 ligase that mediates the ubiquitination of the K48 site of TRAF3, or collects a K48 bit ubiquitinating chain that removes TRAF3 by binding to TRAF3. As a result, we found that the previously reported TRAF3's de ubiquitinase USP25 was not due to I. The addition of OPN is more obvious to remove the ubiquitination chain on TRAF3, and the ubiquitination of K48 in TRAF3 is obviously inhibited by the addition of iOPN. Furthermore, we find that iOPN can obviously inhibit the combination of TRAF3 and Triad3A, and TRAF3 Y440, if the Q442 position is mutated, iOPN can not be combined with the Triad3A. We do not combine with iOPN. It shows that iOPN may be combined with TRAF3 to inhibit the combination of Triad3A and TRAF3, iOPN and Triad3A can be a competitive combination of.7. through the construction of iOPN-WT and iOPN-N end or iOPN-C truncate. IOPN recuperate in the cavity of macrophages, we further confirm that the C end of iOPN is sufficient to play the role of binding TRAF3, inhibiting K48 ubiquitination of TRAF3 and enhancing IFN beta production. It is concluded that the expression of 1.OPN can be induced by the virus, and OPN especially iOPN can regulate I type of interferon in the body as well as type I interferon The strong positive regulator can induce a strong anti-virus immune response,.3. iOPN can regulate the activation of the IRF3 transcription factor,.4. iOPN can be combined with the TRAF3 specificity of the important joint molecule,.5. IOPN can modulate the ubiquitination of TRAF3 K48 bit coupling and make TRAF3 more stable.6. Triad3A mediated, TRAF3 K48 coupled ubiquitination.7. iOPN is combined with TRAF3 through C terminal, and its C end can affect the ubiquitination of TRAF3 as stability. Innovation and significance: 1. this study first clearly and perfectly demonstrated that iOPN can participate in the regulation of antiviral natural immune responses, with positive regulation of the production of IFN beta and obvious The mechanism of this function is that the mechanism of this function is that iOPN inhibits the binding of Triad3A to TRAF3 by inhibiting the ubiquitination of K48 in TRAF3 by Triad3A, and thus leads to the stability of TRAF3, and the activation of IFN beta enhances the.2. based study that the mechanism that iOPN can inhibit the ubiquitination of a molecule is still internationally It is rare, which greatly enriches our understanding of OPN function, and we first found that the C end of iOPN can play almost all the antiviral function.3.. This study provides new experimental evidence for the positive regulation of antiviral immune signaling pathway, which provides new ideas for the research of antiviral and new drug targets.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R392
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本文編號(hào)：1878427