本文選題：單核-巨噬細(xì)胞分化 + caspase-1��； 參考：《南京大學(xué)》2012年博士論文

【摘要】：巨噬細(xì)胞是單核吞噬細(xì)胞系統(tǒng)中的重要成員,在天然免疫和獲得性免疫中都發(fā)揮著重要作用。體內(nèi)巨噬細(xì)胞的來(lái)源包括單核細(xì)胞起源的巨噬細(xì)胞、原位增殖而生成的巨噬細(xì)胞以及卵黃囊巨噬細(xì)胞來(lái)源的巨噬細(xì)胞,其中單核細(xì)胞起源的巨噬細(xì)胞是組織巨噬細(xì)胞的主要來(lái)源。在機(jī)體穩(wěn)態(tài)或發(fā)生炎癥的時(shí)候,單核細(xì)胞都能在微環(huán)境中一些因子的調(diào)控下向巨噬細(xì)胞分化。單核-巨噬細(xì)胞分化過(guò)程的紊亂與多種疾病的發(fā)生發(fā)展密切相關(guān),如自身免疫疾病、自身炎癥性疾病、代謝性疾病以及腫瘤等。因此,對(duì)于單核-巨噬細(xì)胞分化過(guò)程相關(guān)調(diào)控及分子機(jī)制的研究一直以來(lái)都是免疫學(xué)領(lǐng)域的熱點(diǎn)。Caspase-1是caspases家族中與炎癥密切相關(guān)的一個(gè)成員,對(duì)于巨噬細(xì)胞的功能具有關(guān)鍵的調(diào)控作用。Caspase-1缺失的巨噬細(xì)胞釋放炎癥因子IL-1p和IL-18的能力顯著下降;同時(shí)caspase-1還能調(diào)控巨噬細(xì)胞由M2型到M1型的極化。此外,caspase-1也能參與細(xì)胞分化程序的控制。Caspase-1能夠通過(guò)調(diào)控IL-1p的加工、釋放而控制脂肪細(xì)胞和Th17細(xì)胞的分化。但是,對(duì)于caspase-1在單核-巨噬細(xì)胞分化中的功能我們還知之甚少�；赾aspase-1廣泛的生物學(xué)功能及其在單核細(xì)胞、巨噬細(xì)胞的核心地位,caspase-1在單核-巨噬細(xì)胞的分化過(guò)程中也具有重要的調(diào)控功能。在本研究中,我們通過(guò)構(gòu)建體外誘導(dǎo)人單核細(xì)胞白血病細(xì)胞株(THP-1、U937)或人外周血單核細(xì)胞(PBMCs)向巨噬細(xì)胞分化的模型,深入探討了 caspase-1對(duì)于單核-巨噬細(xì)胞分化過(guò)程的調(diào)控效應(yīng)以及所涉及的分子機(jī)理,主要研究?jī)?nèi)容和結(jié)果如下:1.我們運(yùn)用PMA成功誘導(dǎo)THP-1和U937細(xì)胞向巨噬細(xì)胞分化,并通過(guò)分析此過(guò)程中細(xì)胞形態(tài)、表面標(biāo)志分子(markers)及吞噬功能的變化情況而將單核-巨噬細(xì)胞的分化過(guò)程分為早期和晚期兩個(gè)階段。2.我們發(fā)現(xiàn)caspase-1在THP-1和U937細(xì)胞中呈組成型的高表達(dá);然而隨著分化的進(jìn)行其活性不斷升高,并伴隨有IL-1β和IL-18的釋放。Caspase-1活性升高的現(xiàn)象在M-CSF或GM-CSF誘導(dǎo)PBMCs向巨噬細(xì)胞分化的模型中也得到了證實(shí)。并且通過(guò)分析炎癥小體各組分基因表達(dá)結(jié)果顯示caspase-1的活化與炎癥小體的組裝相關(guān)。3. Caspase-1特異性抑制劑WEHD能夠劑量依賴(lài)性地抑制PMA誘導(dǎo)的巨噬細(xì)胞makers (CD11b、CD14)的表達(dá),并且這種抑制作用主要發(fā)生在分化的晚期;同時(shí)WEHD還能明顯抑制巨噬細(xì)胞的吞噬功能。在PBMCs的分化中,WEHD也具有明顯的抑分化效果。4.干擾caspase-1的表達(dá)能抑制caspase-1的激活,并能顯著抑制CD1 1b和CD14的表達(dá)以及細(xì)胞的吞噬活性。我們發(fā)現(xiàn)急性單核細(xì)胞白血病(AML-M5b)病人骨髓單核細(xì)胞中caspase-1的基因水平和蛋白水平均發(fā)生顯著下調(diào),這與AML-M5b中細(xì)胞分化程序的阻滯和單核細(xì)胞的積累密切相關(guān)。5.基于以上的生物學(xué)效應(yīng),我們探討了 caspase-1促進(jìn)單核-巨噬細(xì)胞分化的分子機(jī)制:(1) 在分化早期PPARγ的表達(dá)逐漸升高;而在晚期PPARγ的表達(dá)則明顯下降。Caspase-1的特異性抑制劑或shRNA能夠逆轉(zhuǎn)分化晚期PPARy的下調(diào),但是其mRNA水平卻沒(méi)有明顯改變。在HEK293T細(xì)胞中重組NLRP3炎癥小體能有效激活caspase-1,并能下調(diào)PPARγ的蛋白表達(dá),但caspase-1不能直接切割PPARγ。(2) 在分化早期PPARγ的表達(dá)上調(diào),轉(zhuǎn)錄活性也升高;PPARγ能夠調(diào)控細(xì)胞周期相關(guān)蛋白cyclinD1和p21的表達(dá),從而抑制細(xì)胞周期并促進(jìn)單核-巨噬細(xì)胞的分化進(jìn)程。在分化晚期,PPARy配體troglitazone能夠通過(guò)抑制caspase-1的活性而逆轉(zhuǎn)PPARγ下調(diào)的現(xiàn)象,并誘導(dǎo)NF-κB轉(zhuǎn)錄活性的抑制,從而抑制單核-巨噬細(xì)胞的分化進(jìn)程。(3) 我們還對(duì)caspase-1的經(jīng)典底物在單核-巨噬細(xì)胞分化中的作用進(jìn)行了分析。運(yùn)用washing away、補(bǔ)加細(xì)胞因子或補(bǔ)加細(xì)胞因子對(duì)應(yīng)中和抗體的方法,結(jié)果表明單核-巨噬細(xì)胞的分化不依賴(lài)于IL-1β和IL-18的釋放。IL-1α和IL-33在分化過(guò)程中不釋放,也不具有調(diào)控單核-巨噬細(xì)胞分化的作用。6.通過(guò)構(gòu)建原位乳腺癌種植瘤模型,我們發(fā)現(xiàn)caspase-1抑制劑YVAD能夠誘導(dǎo)腫瘤微環(huán)境中髓源性抑制細(xì)胞(MDSCs)的積累并能促進(jìn)腫瘤的生長(zhǎng),但不影響TAMs的浸潤(rùn)。Caspase-1的活化能夠在體內(nèi)調(diào)控MDSCs的分化。我們的實(shí)驗(yàn)首次闡明了 caspase-1在單核-巨噬細(xì)胞分化中的重要調(diào)控功能;caspase-1能夠通過(guò)下調(diào)晚期分化中PPARγ的表達(dá)而促進(jìn)巨噬細(xì)胞分化的進(jìn)程。并詳細(xì)論述了 PPARγ在巨噬細(xì)胞分化的早期階段和晚期階段發(fā)揮的不同功能。本研究還揭示了 caspase-1的下調(diào)與AML-M5b之間的聯(lián)系,以及caspase-1調(diào)控腫瘤微環(huán)境中MDSCs分化的效應(yīng)。這些發(fā)現(xiàn)表明caspase-1可能成為治療白血病等腫瘤的有效分子靶點(diǎn)。
[Abstract]:Macrophage is an important member of the mononuclear phagocytic cells in the system, in innate and acquired immunity plays an important role. The source of macrophages in vivo including monocyte macrophage origin, generated in situ proliferation of macrophages and macrophages in the yolk sac of macrophage, monocyte macrophage origin which is the main source of tissue macrophages. When body homeostasis or inflammation, mononuclear cells in the microenvironment can control some factors to macrophage differentiation. Closely related to the occurrence and development of mononuclear macrophage differentiation disorder with various diseases, such as autoimmune diseases, autoinflammatory disease, metabolic diseases and cancer. Therefore for the study, mononuclear macrophage differentiation regulation and molecular mechanism of.Ca has been a hotspot in the field of Immunology Spase-1 is a closely related with inflammation of the members of the caspases family, with lack of regulation of.Caspase-1 key for the function of macrophages macrophages to release inflammatory factors IL-1p and IL-18 decreased significantly; at the same time, caspase-1 can also regulate macrophage polarization from type M2 to type M1. In addition,.Caspase-1 caspase-1 can also participate in cell differentiation program through processing the regulation of IL-1p differentiation and control release of fat cells and Th17 cells. However, the function of caspase-1 in monocyte macrophage differentiation are unknown. Based on extensive and biological function of caspase-1 in mononuclear cells, the core position of macrophages, also has an important regulatory function of caspase-1 in monocytes macrophage differentiation. In this study, we constructed an in vitro human monocytic leukemia cell line (T HP-1, U937) or human peripheral blood mononuclear cells (PBMCs) to macrophage differentiation model, discussed the caspase-1 effect for the regulation of monocyte macrophage differentiation and molecular mechanism involved, the main research contents and results are as follows: 1.. We use the PMA THP-1 and U937 cells to induce macrophage differentiation, and through the analysis of cell morphology in this process, the surface marker (markers) and the changes of phagocytosis and differentiation of mononuclear macrophages were divided into early and late two stages of.2. we found that caspase-1 was highly expressed constitutively in THP-1 and U937 cells; however with the differentiation of its activity gradually increased and with the increase of IL-1 beta and IL-18 release of.Caspase-1 activity induced by PBMCs in M-CSF or GM-CSF phenomenon to macrophage differentiation model has also been confirmed. And through the analysis of inflammation Body components of gene expression showed that caspase-1 inflammasome activation and assembly of.3. Caspase-1 specific inhibitor WEHD dose dependently inhibited PMA induced macrophage makers (CD11b, CD14) expression, and this inhibition occurs mainly in the differentiation of the advanced stage; while WEHD can obviously inhibit the phagocytosis of macrophages. The differentiation of PBMCs, WEHD also has the effect of.4. interference inhibiting the expression of differentiation caspase-1 can obviously inhibit the activity of Caspase-1, and the expression of CD1 and CD14 can significantly inhibit 1b and phagocytic activity. We found that acute monocytic leukemia (AML-M5b) patients with caspase-1 bone marrow mononuclear cells in gene and protein levels there were significantly reduced, and the arrest of cell differentiation in the AML-M5b program and the accumulation of monocytes is closely related to the biological effects of.5. on the basis of the above, I We discussed the molecular mechanism of Caspase-1 promoting monocyte macrophage differentiation: (1) the expression of PPAR increased gradually during the early differentiation of gamma; expression in advanced PPAR gamma decreased the specific inhibitor of.Caspase-1 or shRNA can down regulate the differentiation of reversal of advanced PPARy, but the mRNA level did not changed obviously in HEK293T cells. Recombinant NLRP3 inflammasome can effectively activate caspase-1, and down regulate the expression of PPAR gamma protein, but caspase-1 cannot cut PPAR gamma directly. (2) raised in the early differentiation of PPAR gamma expression and transcriptional activity also increased; PPAR expression can regulate cell cycle related proteins cyclinD1 and p21, and inhibit the cell cycle and promote monocyte macrophage differentiation process. In the late differentiation stage, PPARy ligand troglitazone can inhibit caspase-1 activity and reverse PPAR gamma cut, and induce NF- kappa B transcription activity Inhibition inhibits monocyte macrophage differentiation process. (3) we are also on the classic caspase-1 substrate on monocyte macrophage differentiation were analyzed. Using washing away method, adding additional cytokines or cytokine corresponding neutralizing antibodies, the results show that the differentiation of mononuclear macrophage not depending on the IL-1 beta and IL-18 release of.IL-1 alpha and IL-33 was not released in the process of differentiation,.6. also has no regulation of monocyte macrophage differentiation by constructing tumor model of breast cancer in situ cultivation, we found that Caspase-1 inhibitor YVAD can induce tumor myeloid derived suppressor cells in the microenvironment (MDSCs) accumulation and to promote tumor growth, but does not affect the differentiation of TAMs activation in vivo invasion of.Caspase-1 can regulate MDSCs. Our experiments for the first time to clarify the important caspase-1 on monocyte macrophage differentiation in control Function; caspase-1 can promote macrophage differentiation process through the expression of differentiation in the late stage of PPAR gamma. And discusses the different functions of PPAR gamma in macrophage differentiation in the early stage and late stage play. This study also reveals that the downregulation of caspase-1 and AML-M5b between the contact and the regulation effect of caspase-1 in tumor microenvironment MDSCs differentiation. These findings suggest that caspase-1 may be an effective molecular target for the treatment of leukemia and other tumors.

【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 李小穎;馬元武;張旭;張連峰;;不同接種量熒光素酶標(biāo)記小鼠乳腺癌細(xì)胞4T1在小鼠體內(nèi)生長(zhǎng)及肺轉(zhuǎn)移的比較[J];中國(guó)實(shí)驗(yàn)動(dòng)物學(xué)報(bào);2012年01期

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本文編號(hào)：1746047