發(fā)布時間：2018-05-12 11:23

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

【摘要】：固有免疫系統作為機體抵御外界病原體入侵的首道防線,不僅可以激活適應性免疫系統,同時也可以直接對入侵的病原體產生強烈的免疫應答,從而殺傷病原體。在病毒入侵時,固有免疫系統可以激活產生多種細胞因子,其中最主要的是I型干擾素(IFNα/β)。固有免疫系統中的多種的模式識別受體(PRRs)都與Ⅰ型干擾素的產生相關,包括諸如Toll樣受體(TLRs), RIG-I樣受體(RLRs)和內源性DNA感受器等。產生的Ⅰ型干擾素可以進而與其受體結合,活化JAK (Janus kinase)和STAT (Signaltransducers and activators of transcription)信號通路,進而誘導IFN-stimulated genes (ISGs)的表達,最終清除病毒。骨橋蛋白(osteopontin, OPN)又稱為早期T淋巴細胞活化因子1(early T lymphocyte activation 1, Etal),是一種分泌性的多功能糖蛋白。OPN可以調節(jié)多種生物體進程,包括細胞分化、粘附、骨重構、惡性腫瘤和免疫反應。長期以來,OPN被認為是一個與炎癥過程有關的潛在的促炎癥細胞因子,具有促進巨噬細胞分泌IFN-γ和IL-12的作用。近年來,隨著胞內形式OPN (iOPN)的發(fā)現,OPN在不同免疫細胞以及免疫反應的不同階段中的不同調控作用逐漸引起人們的重視。而iOPN在固有免疫系統當中尤其是抗病毒免疫當中的作用還未明確。研究目的：探討OPN,尤其是iOPN是否可以調節(jié)病毒誘導的Ⅰ型干擾素產生。如若可以調節(jié)Ⅰ型干擾素產生,闡述其潛在機制。研究方法：1.首先檢測不同病毒及刺激劑對OPN表達的影響。再利用OPN缺陷型(Spp1-/-)小鼠與野生型(WT)小鼠的腹腔巨噬細胞,經病毒刺激后,檢測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。通過構建全長型OPN (full length OPN)及胞內形式OPN(iOPN)表達質粒并轉染進HEK293細胞,利用雙熒光素酶報告基因,檢測兩種不同形式OPN對于調節(jié)RNA病毒SeV所活化的IFN表達的影響,并檢測iOPN質粒對于各種接頭分子所活化的IFN啟動子區(qū)活性的調節(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VSV病毒在小鼠臟器中的滴度與含量,反應兩類小鼠對于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)小鼠腹腔巨噬細胞,以及iOPN過表達質粒轉染HEK293細胞,檢測經SeV刺激后,重要的IFN轉錄因子IRF3的活化水平變化。4.經實時熒光定量PCR (q-PCR)以及雙熒光素酶報告基因實驗,檢測iOPN質粒的靶點分子,并經免疫共沉淀(co-IP)以及免疫熒光等實驗檢測iOPN與靶點分子的結合情況。5.明確靶點分子后,利用不同類型的泛素分子質粒,共轉染iOPN及靶點分子進HEK293細胞,檢測其是否發(fā)生泛素化水平的變化,以及靶點分子的穩(wěn)定性是否發(fā)生變化。6.靶點分子若發(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可能可以調節(jié)靶點分子的穩(wěn)定性。通過對靶點已知的泛素化酶以及去泛素化酶進行篩選,尋找可能的作用機制,并通過細胞水平以及體外翻譯系統對結論進行實驗性確認。7.通過利用細胞過表達載體以及慢病毒表達載體構建iOPN-WT、iOPN-N、iOPN-C重組過表達載體,通過體外翻譯系統實驗或對OPN缺陷型小鼠腹腔巨噬細胞進行iOPN回補實驗,明確iOPN具體發(fā)生功能的區(qū)域。研究結果：1.首先,在利用RNA病毒VSV(水泡性口炎病毒)、SeV(仙臺病毒)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胞不同時間點后,發(fā)現OPN的表達都有逐漸升高的趨勢；其次,在OPN缺陷型小鼠以及野生型小鼠巨噬細胞中加入SeV或VSV刺激不同時間后,發(fā)現相比較于野生型組,OPN缺陷型小鼠腹腔巨噬細胞產生IFNβ的能力都明顯降低；將構建成功的全長型OPN表達載體與iOPN表達載體分別轉染進HEK293后經SeV刺激后,發(fā)現二種質粒都可以使IFNβ表達增強,但是iOPN表達載體組明顯強于全長型OPN表達載體組,并且利用OPN封閉抗體封閉分泌型OPN后,IFNβ的產生無明顯變化,說明全長型OPN所表達的分泌性OPN可能并不參與調節(jié)IFNβ的增強過程；iOPN也可以明顯增強RIG-I、MDA5、TRIF以及STINGcGAS等接頭分子所介導的IFNβ啟動子區(qū)的活化。2.OPN缺陷型小鼠相比較于野生型小鼠,會產生較少的IFNβ,進而造成此組巨噬細胞或成年小鼠在經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.前述實驗結果證明OPN,尤其是iOPN的確可以正調IFNβ的產生,又由于IRF3是重要的調控IFNβ表達的轉錄因子,我們進而研究IRF3的活化水平是否有變化。我們發(fā)現無論是在啟動子區(qū)結合、磷酸化、二聚化以及入核等方面,iOPN都可以起到正調作用,說明iOPN的確是通過影響IRF3轉錄因子的活化進而增強了IFNβ的表達。4.為了明確iOPN作用的靶點,我們經報告基因以及realtime-PCR等技術發(fā)現iOPN作用的靶點可能存在于MAVS以及TBKl附近。通過免疫共沉淀我們發(fā)現iOPN可以特異性與TRAF3結合,并且這一結論也經免疫熒光以及體外蛋白結合實驗驗證。5.在知道作用靶點為TRAF3后,我們進一步研究可能的機制。泛素化調節(jié)作用在機體中具有舉足輕重的作用,我們首先研究下iOPN是否會影響TRAF3的泛素化。結果發(fā)現,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表達的作用。無論是在HEK293細胞中過表達iOPN,或是在OPN缺陷型的小鼠腹腔巨噬細胞,iOPN的過表達或缺失都會導致放線菌酮(CHX)介導的TRAF3的穩(wěn)定性發(fā)生明顯變化。6.由于我們發(fā)現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TRAF3的K48位泛素化作用的E3連接酶與其結合,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結合,結果我們發(fā)現之前有報道過的TRAF3的去泛素化酶USP25并沒有因為iOPN的加入而更加明顯的除去TRAF3上的泛素化鏈,而Triad3A對于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。進而我們發(fā)現iOPN可以明顯的抑制TRAF3與Triad3A的結合,并且TRAF3的Y440、Q442位點如若被突變則TRAF3不與Triad3A結合,同時也不與iOPN結合。綜上說明iOPN可能是通過結合了TRAF3進而抑制了Triad3A與TRAF3的結合,iOPN與Triad3A肯能是競爭結合關系。7.通過構建的iOPN-WT以及iOPN-N端或iOPN-C端截短體,我們發(fā)現iOPN的C端可以與TRAF3發(fā)生結合,并且通過在OPN缺陷型小鼠腹腔巨噬細胞中進行iOPN回補實驗,我們進一步確定iOPN的C端即足以發(fā)揮結合TRAF3、抑制TRAF3的K48位泛素化以及增強IFNβ產生的作用�？偨Y：1.OPN的表達可被病毒所誘導,并且OPN尤其是iOPN可以正調I型干擾素的產生。2.OPN在體內也是Ⅰ型干擾素產生的強力正調者,可以誘導很強的抗病毒免疫應答。3. iOPN可以正調IRF3轉錄因子的活化。4. iOPN可以與重要接頭分子TRAF3特異性結合。5. IOPN可以調節(jié)TRAF3的K48位偶聯的泛素化修飾并且使TRAF3更穩(wěn)定。6. iOPN與Triad3A競爭性結合TRAF3,進而影響Triad3A介導的,TRAF3的K48位偶聯的泛素化。7. iOPN通過C端與TRAF3結合,并且其C端即可影響TRAF3的泛素化即穩(wěn)定性。創(chuàng)新點及意義：1.本研究首次明確完善地證明iOPN可以參與調控抗病毒天然免疫反應,可以正向調節(jié)IFNβ的產生以及具有明顯的抗病毒功能。這一功能的機制是由于iOPN通過抑制Triad3A與TRAF3的結合,從而抑制了Triad3A對TRAF3的K48位泛素化修飾作用,進而導致TRAF3的穩(wěn)定性增強,IFNβ的活化進而增強。2.本研究提出iOPN可以抑制某分子的泛素化作用這一機制在國際上尚屬少見,極大豐富了我們對于OPN功能的理解。并且我們首次發(fā)現iOPN的C端可以發(fā)揮幾乎全部的抗病毒功能。3.本研究為正向調控抗病毒免疫信號通路提供了新的實驗證據,為抗病毒研究以及新藥物靶點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.

【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R392
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本文編號：1878427