【摘要】：天然免疫應(yīng)答是機(jī)體抵抗病原微生物入侵的第一道防線,但是,過(guò)度活化的天然免疫反應(yīng)會(huì)導(dǎo)致組織器官的損傷,可導(dǎo)致自身免疫病的產(chǎn)生甚至造成病人死亡,所以,天然免疫應(yīng)答反應(yīng)需要及時(shí)活化以有效清除入侵的病原體,同時(shí),其應(yīng)答程度與持續(xù)時(shí)間也應(yīng)控制在適度范圍內(nèi)以維持機(jī)體平衡,可見(jiàn),天然免疫應(yīng)答反應(yīng)的負(fù)向調(diào)控對(duì)于機(jī)體維持免疫自穩(wěn)至關(guān)重要。近年來(lái),發(fā)現(xiàn)與研究能夠負(fù)向調(diào)控天然免疫的細(xì)胞和分子逐步成為天然免疫研究領(lǐng)域的前沿?zé)狳c(diǎn)。本課題圍繞著兩個(gè)負(fù)向調(diào)控分子的作用與相關(guān)機(jī)制,分兩部分開(kāi)展了負(fù)向調(diào)控天然免疫應(yīng)答及其機(jī)制研究,包括"LAPF抑制TLR觸發(fā)巨噬細(xì)胞產(chǎn)生TNF-α及其機(jī)制研究”和"CD11b-Src信號(hào)通路促進(jìn)M2型巨噬細(xì)胞產(chǎn)生及其機(jī)制研究”,以期通過(guò)我們的研究,加深對(duì)天然免疫調(diào)節(jié)和免疫自穩(wěn)的認(rèn)識(shí)和理解。第一部分LAPF抑制TLR觸發(fā)巨噬細(xì)胞產(chǎn)生TNF-a及其機(jī)制研究包含PH和FYVE結(jié)構(gòu)域的溶酶體相關(guān)凋亡誘導(dǎo)蛋白LAPF(又叫PLEKHF1)是我們實(shí)驗(yàn)室從人樹(shù)突狀細(xì)胞cDNA文庫(kù)中通過(guò)隨機(jī)大規(guī)模測(cè)序發(fā)現(xiàn)的一個(gè)新的溶酶體相關(guān)蛋白并于2005年報(bào)道其參與TNF-a誘導(dǎo)的腫瘤細(xì)胞凋亡。然而有關(guān)LAPF在天然免疫中的功能還沒(méi)有研究。在本課題中,我們制備了LAPF條件缺失的小鼠( lapfloxp/loxplyz2Cre/+),發(fā)現(xiàn)LAPF條件缺失小鼠相比于雜合子對(duì)照小鼠在內(nèi)毒素休克模型中炎性細(xì)胞因子分泌增加,其中TNF-α顯著升高,同時(shí)肺臟和脾臟中TNF-α的mRNA水平也顯著升高,提示LAPF可能負(fù)向調(diào)控天然免疫反應(yīng)與炎癥發(fā)生。通過(guò)體外細(xì)胞實(shí)驗(yàn),發(fā)現(xiàn)LAPF缺失的巨噬細(xì)胞在TLR配體刺激下分泌TNF-α升高并且TAK-、 IKKa/β和p65的磷酸化活化水平顯著提高,表明LAPF能夠抑制NF-κB信號(hào)通路的活化。利用Ip-質(zhì)譜分析結(jié)合體外IP驗(yàn)證,我們發(fā)現(xiàn)LAPF可以與TLR信號(hào)關(guān)鍵轉(zhuǎn)運(yùn)蛋白TIRAP和MyD88結(jié)合,為進(jìn)一步研究LAPF如何調(diào)控NF-κB信號(hào)通路、影響炎性細(xì)胞因子釋放奠定了基礎(chǔ)。第二部分CDllb-Src信號(hào)通路促進(jìn)M2型巨噬細(xì)胞產(chǎn)生及其機(jī)制研究整合素信號(hào)對(duì)于天然免疫應(yīng)答的調(diào)控一直是天然免疫的前沿?zé)狳c(diǎn)問(wèn)題。整合素對(duì)于天然免疫細(xì)胞的分化、發(fā)育、遷移、粘附和活化均有十分重要的作用。CDllb是整合素Mac-1的a鏈,CDllb的抗體常常用來(lái)篩選出巨噬細(xì)胞或者其它髓系來(lái)源的單核吞噬細(xì)胞。巨噬細(xì)胞根據(jù)表型和行使功能的不同又可以分為M1型(經(jīng)典性活化的)巨噬細(xì)胞和M2型(替代性活化的)巨噬細(xì)胞,分別釋放出促炎性細(xì)胞因子和抑炎性細(xì)胞因子,M1和M2型巨噬細(xì)胞的平衡對(duì)于天然免疫的調(diào)節(jié)和自穩(wěn)至關(guān)重要。我們實(shí)驗(yàn)室以往發(fā)現(xiàn)CDllb能夠通過(guò)Src-Syk泛素化降解MyD88和TRIF,從而抑制天然免疫過(guò)程中TNF-a的產(chǎn)生。但是,CD11b-Src在巨噬細(xì)胞M1和M2的表型和功能中的作用并不清楚,值得進(jìn)一步研究。我們發(fā)現(xiàn)在體外誘導(dǎo)腸炎模型中,CD1lb缺陷的小鼠以及利用Src抑制劑Dasatinib腹腔注射處理的小鼠,結(jié)腸部位的炎癥反應(yīng)均更為嚴(yán)重,小鼠TNF-a產(chǎn)量增加但I(xiàn)L-10的產(chǎn)量減少,同時(shí),iNOS的表達(dá)增強(qiáng)但Arg-1的表達(dá)減弱。TNF-a和iNOS是M1型巨噬細(xì)胞的標(biāo)志性分子,IL-10和Arg1是M2型巨噬細(xì)胞的標(biāo)志性分子,這個(gè)現(xiàn)象提示了CD11b-Src信號(hào)可能促使巨噬細(xì)胞趨向于M2型巨噬細(xì)胞。為此,我們直接檢測(cè)了用Dasatinib處理并且誘導(dǎo)腸炎的小鼠結(jié)腸部位的巨噬細(xì)胞類型,發(fā)現(xiàn)M1型巨噬細(xì)胞比例增加、M2型巨噬細(xì)胞比例減少。隨后我們開(kāi)展了CD11b-Src使巨噬細(xì)胞更趨向于M2型巨噬細(xì)胞相關(guān)機(jī)制研究,發(fā)現(xiàn)1)骨髓來(lái)源的巨噬細(xì)胞中CD11b-Src可以抑制IFN-γ誘導(dǎo)的M1型巨噬細(xì)胞標(biāo)志分子iNOS的表達(dá),CD11b-Src還能夠促進(jìn)IL-4誘導(dǎo)的M2型巨噬細(xì)胞標(biāo)志分子Arg-1和YM-1的表達(dá)；2)CDllb通過(guò)促進(jìn)Src-Akt信號(hào)通路活化,促進(jìn)了TLR誘導(dǎo)的STAT6活化和IL-10釋放。進(jìn)一步的機(jī)制研究發(fā)現(xiàn),Src能夠增強(qiáng)IL-4信號(hào)通路的Akt和STAT6活化,這兩者均被報(bào)道能夠促進(jìn)M2型巨噬細(xì)胞表型分子的表達(dá)。但是,只有過(guò)表達(dá)STAT6能夠逆轉(zhuǎn)Src抑制劑對(duì)Argl的下調(diào)作用,提示STAT6在此過(guò)程的重要作用。Src能夠和STAT6相互作用,形成復(fù)合體,也進(jìn)一步提示Src能夠參與活化STAT6.在TLR信號(hào)刺激下,Src則通過(guò)促進(jìn)P13K的調(diào)節(jié)亞基p85的泛素化降解,促進(jìn)PI3K-Akt信號(hào)通路活化,進(jìn)而通過(guò)負(fù)向調(diào)控GSK促進(jìn)AP-1的活化入核,最終促進(jìn)IL-10的產(chǎn)生。綜上,CD11b-Src通過(guò)促進(jìn)STAT6和Akt活化而使巨噬細(xì)胞更趨向于M2型巨噬細(xì)胞。可見(jiàn),CD11b-Src具有重要的天然免疫負(fù)向調(diào)控與控制炎癥的作用,深入研究CD11b-Src的機(jī)制將有助于深入認(rèn)識(shí)炎癥的發(fā)生發(fā)展過(guò)程和抗炎藥物的研發(fā)。
[Abstract]:The natural immune response is the first line of defense for the organism to resist the invasion of pathogenic microbes. However, the excessive activation of natural immune response can lead to the injury of the tissues and organs, which can lead to the production of autoimmune diseases and even cause the death of the patients. Therefore, the natural immune response should be activated in time to effectively remove the invading pathogens. The degree and duration of answer should also be controlled in a moderate range to maintain the balance of the body. It can be seen that the negative regulation of natural immune response is crucial to the maintenance of immune self stability. In recent years, the discovery and study of cells and molecules that can negatively regulate natural immunity gradually become the forefront of the field of natural immune research. Around the role of two negative regulators and related mechanisms, two parts of the negative regulation of natural immune response and its mechanism are carried out, including "LAPF inhibition of TLR triggering macrophages to produce TNF- alpha and its mechanism research" and "CD11b-Src signaling pathway to promote the production and mechanism of M2 macrophages", with a view to through our research To deepen the understanding and understanding of natural immune regulation and immune self stability. Part 1 LAPF inhibits TLR triggering macrophages to produce TNF-a and its mechanism, which includes PH and FYVE domain lysosome related apoptosis induced protein LAPF (also called PLEKHF1) which is by random large-scale sequencing from human dendritic cell cDNA library. A new lysosome related protein is now reported to be involved in the apoptosis of tumor cells induced by TNF-a in 2005. However, the function of LAPF in natural immunity has not been studied. In this subject, we prepared the LAPF deficient mice (lapfloxp/loxplyz2Cre/+), and found that the LAPF condition deficient mice were compared to the heterozygote control mice. The secretion of inflammatory cytokines increased in the endotoxin shock model, in which TNF- alpha was increased significantly, while the mRNA level of TNF- a in the lungs and spleen also increased significantly, suggesting that LAPF may negatively regulate the natural immune response and inflammation. Through in vitro cell experiments, it was found that the LAPF deficient macrophages secreted the increase of TNF- alpha under the TLR ligand stimulation. And TAK-, the activation level of phosphorylation of IKKa/ beta and p65 significantly increased, indicating that LAPF could inhibit the activation of NF- kappa B signaling pathway. Using Ip- mass spectrometry analysis and in vitro IP validation, we found that LAPF can be combined with TIRAP and MyD88 with TLR signal key transporter protein, which can further study the regulation of inflammatory cells and affect inflammatory cells. Factor release lays the foundation. Second CDllb-Src signaling pathway promotes the production and mechanism of M2 type macrophages. The regulation of integrin signal for natural immune response has been a hot issue in the frontier of natural immunity. Integrin has a very important role in the differentiation, development, migration, adhesion and activation of natural immune cells. CDllb is the a chain of integrin Mac-1, and CDllb antibodies are often used to screen out macrophages or other myeloid mononuclear phagocytes. Macrophages can be divided into M1 type (classic activated) macrophages and M2 type macrophages based on the phenotype and function of the macrophages, which release proinflammatory cytokines, respectively. The balance between M1 and M2 macrophages is essential for the regulation and self stability of natural immunity. In our laboratory, we have found that CDllb can degrade MyD88 and TRIF through Src-Syk ubiquitination, thus inhibiting the production of TNF-a in natural immune processes. However, CD11b-Src is in the phenotype and function of M1 and M2 in macrophages. The effect was not clear and worthy of further study. In the model of in vitro induced enteritis, we found that the CD1lb deficient mice and the mice treated with the Src inhibitor Dasatinib intraperitoneally were more severe in the colon, and the production of TNF-a in the mice was increased but the production of IL-10 decreased, and the expression of iNOS was enhanced but the expression of Arg-1 was enhanced. Weakened.TNF-a and iNOS are marker molecules of M1 type macrophages, IL-10 and Arg1 are marker molecules of M2 macrophages. This phenomenon suggests that CD11b-Src signals may induce macrophages to tend to M2 type macrophages. To this end, we directly detected the macrophages in the colon of mice that were treated with Dasatinib and induced enteritis. The proportion of M1 type macrophages increased and the proportion of M2 type macrophages decreased. Then we carried out CD11b-Src to make macrophages more inclined to M2 macrophage related mechanisms, and found that CD11b-Src in bone marrow derived macrophages can inhibit the expression of the M1 type macrophage marker molecule iNOS, which is induced by IFN- gamma, and CD11b-Src also can be used. Promote the expression of IL-4 induced M2 macrophage markers Arg-1 and YM-1; 2) CDllb promotes TLR induced STAT6 activation and IL-10 release by promoting the activation of Src-Akt signaling pathway. Further mechanism studies found that Src enhances Akt and activation of IL-4 signaling pathways, both of which are reported to promote macrophages. The expression of phenotypic molecules. However, only overexpression of STAT6 can reverse the downregulation of Src inhibitors to Argl, suggesting the important role of STAT6 in this process,.Src can interact with STAT6 to form a complex, and further hints that Src can participate in activated STAT6. in the TLR signal stimulation, Src by promoting P13K to regulate the ubiquitin of subunit p85. Degradation, promoting activation of PI3K-Akt signaling pathway, and further promoting the activation of AP-1 through negative regulation of GSK, eventually promotes the production of IL-10. In conclusion, CD11b-Src makes macrophages more likely to be M2 type macrophages by promoting STAT6 and Akt activation. Thus, CD11b-Src has an important role in natural immune negative regulation and control of inflammation. Studying the mechanism of CD11b-Src will help us to further understand the occurrence and development of inflammation and the research and development of anti-inflammatory drugs.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【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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