本文選題：GDF15 + 巨噬細(xì)胞��； 參考：《第四軍醫(yī)大學(xué)》2016年博士論文

【摘要】：全球范圍內(nèi)惡性腫瘤的發(fā)病率仍將處于不斷增長(zhǎng)的趨勢(shì)。近年來(lái),腫瘤免疫治療因其良好的臨床療效,得到越來(lái)越多的重視和認(rèn)可。然而,腫瘤免疫逃逸嚴(yán)重限制了腫瘤免疫治療的效果。研究表明,腫瘤微環(huán)境下免疫細(xì)胞的功能失調(diào)在腫瘤免疫逃逸過(guò)程中發(fā)揮重要作用。巨噬細(xì)胞是固有免疫系統(tǒng)中重要的細(xì)胞組成部分,在抗感染、提呈抗原啟動(dòng)免疫應(yīng)答、抗腫瘤及免疫調(diào)節(jié)過(guò)程中發(fā)揮重要作用。在不同微環(huán)境的影響下,巨噬細(xì)胞分化為不同的表型并表現(xiàn)出不同的功能,即極化現(xiàn)象。目前將巨噬細(xì)胞主要分為兩大類(lèi),即以分泌促炎因子為主的經(jīng)典活化M1型巨噬細(xì)胞和以發(fā)揮消炎反應(yīng)、促進(jìn)組織修復(fù)功能為主的替代性活化M2型巨噬細(xì)胞。其中,M1型巨噬細(xì)胞可誘導(dǎo)Th1反應(yīng)的發(fā)生,促進(jìn)炎性因子(如IL-12等)介導(dǎo)的Th1細(xì)胞應(yīng)答,殺傷胞內(nèi)寄生菌和腫瘤細(xì)胞;M2型巨噬細(xì)胞通過(guò)支持Th2細(xì)胞相關(guān)的效應(yīng)功能,在抑制機(jī)體免疫系統(tǒng)殺傷腫瘤細(xì)胞的過(guò)程中發(fā)揮作用。大量的臨床和實(shí)驗(yàn)證據(jù)表明,在大多數(shù)腫瘤中,腫瘤組織浸潤(rùn)的巨噬細(xì)胞,即腫瘤相關(guān)巨噬細(xì)胞,普遍具有m2樣表型,可分泌多種生長(zhǎng)因子促進(jìn)腫瘤血管生成,分泌多種參與基質(zhì)降解的酶類(lèi)促進(jìn)腫瘤的侵襲和轉(zhuǎn)移,誘導(dǎo)treg細(xì)胞分化進(jìn)而抑制cd8+t細(xì)胞的細(xì)胞毒性,抑制ctl細(xì)胞和nk細(xì)胞的增殖和殺傷功能、抑制dc細(xì)胞的活化、成熟和抗原提呈功能,從而參與腫瘤免疫逃逸,促進(jìn)腫瘤的惡性發(fā)展。gdf15首次從人單核細(xì)胞系u937的巨噬細(xì)胞活化相關(guān)基因cdna文庫(kù)中鑒定并表達(dá),是tgf-β超家族的成員,參與了多種生理和病理過(guò)程。gdf15的表達(dá)失調(diào)還與多種腫瘤的發(fā)生發(fā)展密切相關(guān),并直接影響患者的預(yù)后和生存質(zhì)量。雖然gdf15作為腫瘤源性因子在腫瘤的發(fā)生發(fā)展過(guò)程中的作用已有較多關(guān)注,但時(shí)至今日,關(guān)于gdf15對(duì)免疫細(xì)胞作用的明確報(bào)道依然廖廖無(wú)幾。我們前期研究了gdf15對(duì)dc及treg細(xì)胞分化成熟和功能的影響,首次證實(shí)gdf15不僅在dc的成熟和功能發(fā)揮過(guò)程中發(fā)揮顯著的抑制效應(yīng),還可以通過(guò)上調(diào)foxp3表達(dá),促進(jìn)naivecd4+t細(xì)胞向treg分化成熟。那么,gdf15是否可影響巨噬細(xì)胞的極化?其誘導(dǎo)巨噬細(xì)胞發(fā)揮免疫抑制功能的機(jī)制又是什么?為闡明上述問(wèn)題,我們進(jìn)行了以下研究工作:1)首先從人外周血單個(gè)核細(xì)胞中分離得到cd14+單核細(xì)胞,進(jìn)一步誘導(dǎo)獲得巨噬細(xì)胞。通過(guò)real-timepcr、elisa和fcm等方法研究gdf15對(duì)cd14+單核細(xì)胞及其來(lái)源巨噬細(xì)胞極化階段膜分子表達(dá)、細(xì)胞因子分泌、吞噬能力及誘導(dǎo)na?vecd4+t細(xì)胞表達(dá)foxp3能力的影響;2)利用慢病毒感染構(gòu)建gdf15過(guò)表達(dá)的小鼠結(jié)腸癌ct26穩(wěn)轉(zhuǎn)細(xì)胞系;3)通過(guò)real-timepcr、fcm和mlr等方法研究gdf15對(duì)鼠髓源性巨噬細(xì)胞及其極化階段膜分子表達(dá)、細(xì)胞因子分泌、no分泌及刺激t細(xì)胞增殖能力的影響;4)構(gòu)建荷瘤小鼠,通過(guò)elisa、real-timepcr和fcm等方法研究gdf15的體內(nèi)促瘤作用及對(duì)tam的影響;5)選取thp-1細(xì)胞作為人巨噬細(xì)胞的模式細(xì)胞,通過(guò)real-timepcr、elisa和fcm等方法驗(yàn)證gdf15對(duì)thp-1細(xì)胞及其極化階段膜分子表達(dá)和細(xì)胞因子分泌的影響,并選擇其中受gdf15影響最顯著的tgf-β1,進(jìn)一步通過(guò)雙熒光素酶報(bào)告基因系統(tǒng)、real-timepcr、elisa、westernblot、轉(zhuǎn)錄因子活性芯片、激光共聚焦、chip和rna干涉等方法研究gdf15調(diào)控tgf-β1表達(dá)的分子機(jī)制。通過(guò)以上研究工作,我們得到了如下結(jié)果:1)gdf15直接誘導(dǎo)cd14+單核細(xì)胞向m2樣巨噬細(xì)胞極化,在cd14+單核細(xì)胞來(lái)源巨噬細(xì)胞極化階段可抑制m1型巨噬細(xì)胞極化,誘導(dǎo)m2樣巨噬細(xì)胞極化;2)gdf15刺激后的m2樣巨噬細(xì)胞能夠誘導(dǎo)Treg形成;3)GDF15可顯著誘導(dǎo)CD14+單核細(xì)胞及其來(lái)源巨噬細(xì)胞在極化階段表達(dá)TGF-β1;4)GDF15可誘導(dǎo)鼠髓源性巨噬細(xì)胞及其極化階段向M2樣巨噬細(xì)胞極化,并抑制其刺激T細(xì)胞增殖的能力;5)過(guò)表達(dá)GDF15可促進(jìn)腫瘤體內(nèi)生長(zhǎng),增加腹腔M2樣巨噬細(xì)胞比率及M2樣TAM浸潤(rùn);6)血漿GDF15水平、CD206+TAM浸潤(rùn)比率及TAM的TGF-β1表達(dá)水平,三者之間成兩兩正相關(guān);7)GDF15可誘導(dǎo)THP-1細(xì)胞及其極化階段表達(dá)TGF-β1;8)GDF15通過(guò)K-Ras-JNK/ERK-c-Myc通路調(diào)控TGF-β1表達(dá)。我們首次證實(shí)GDF15可誘導(dǎo)巨噬細(xì)胞產(chǎn)生M2樣表型和功能;GDF15抑制巨噬細(xì)胞向M1型極化,誘導(dǎo)其向M2樣極化;GDF15顯著誘導(dǎo)巨噬細(xì)胞及其極化階段表達(dá)免疫抑制性因子TGF-β1;GDF15在CT26細(xì)胞過(guò)表達(dá)可提高其成瘤活性。為闡明GDF15如何誘導(dǎo)巨噬細(xì)胞表達(dá)TGF-β1,我們對(duì)相關(guān)機(jī)制進(jìn)行研究,從而為腫瘤免疫耐受的發(fā)生提供新的研究方向,為阻斷腫瘤免疫逃逸、提高腫瘤免疫治療效果提供新思路和新靶點(diǎn)。
[Abstract]:The incidence of malignant tumor in the world is still in the growing trend. In recent years, tumor immunotherapy has gained more and more attention and recognition because of its good clinical effect. However, tumor immune escape seriously restricts the effect of tumor immunotherapy. Macrophages are important cellular components in the immune system. Macrophages play an important role in anti infection, antigen activated immune response, anti-tumor and immunomodulatory processes. Under the influence of different microenvironments, macrophages are differentiated into different phenotypes and exhibit different functions. At present, the macrophage is divided into two major categories: the classic activated M1 macrophages which mainly secrete proinflammatory factors and the alternative activated M2 macrophages which play an inflammatory response to promote the tissue repair function. Among them, M1 type macrophages can induce the occurrence of Th1 reaction and promote inflammatory factors (such as IL-12, etc.). Th1 cell response, killing intracellular parasites and tumor cells; M2 macrophages play a role in inhibiting the killing of tumor cells in the immune system by supporting Th2 cell related effects. A large number of clinical and experimental evidence suggests that in most tumors, the macrophages infiltrated by tumor tissue, that is, tumor related Macrophages, which generally have M2 like phenotype, can secrete a variety of growth factors to promote tumor angiogenesis, secrete a variety of enzymes involved in matrix degradation to promote tumor invasion and metastasis, induce Treg cells to differentiate and then inhibit the cytotoxicity of cd8+t cells, inhibit the proliferation and killing function of CTL cells and NK cells, and inhibit the activation of DC cells. Maturation and antigen presentation function, thus participating in tumor immune escape and promoting the malignant development of tumor,.Gdf15 is first identified and expressed from the macrophage activation related gene cDNA library of human mononuclear cell line U937. It is a member of the tgf- beta superfamily, and participates in a variety of physiological and pathological processes of.Gdf15 and the occurrence of many kinds of tumors. It is closely related and has a direct impact on the prognosis and quality of life. Although GDF15 has been paid more attention to the role of tumor derived factors in the development of tumor, there are still few reports about the role of GDF15 in immune cells. We studied the differentiation and maturation of DC and Treg cells by GDF15 in the previous period. The effect of function is the first confirmation that GDF15 not only plays a significant inhibitory effect in the process of maturation and function of DC, but also can promote the differentiation and maturation of naivecd4+t cells to Treg by up regulation of Foxp3 expression. Then, does GDF15 affect the polarization of macrophages? What is the mechanism of inducing macrophage cells to play an immunosuppressive function? In order to clarify the above problems, we have carried out the following research work: 1) first, cd14+ mononuclear cells were isolated from human peripheral blood mononuclear cells to further induce macrophages. Through real-timepcr, ELISA and FCM methods, the expression of GDF15 on the membrane molecules in the polarization phase of cd14+ mononuclear cells and their source macrophage cells and cytokine secretion were studied. The effect of phagocytosis and induction of the expression of Foxp3 in Na vecd4+t cells; 2) using the lentivirus infection to construct GDF15 overexpressed mouse colon cancer CT26 stable cell line; 3) the expression of membrane molecules, cytokine secretion, NO secreting and stimulating T cells in GDF15 mouse medullary macrophages and its polarization phase were studied by real-timepcr, FCM and MLR. The effect of proliferation ability; 4) the tumor bearing mice were constructed, the tumor promoting effect of GDF15 in vivo and the effect on TAM were studied by ELISA, real-timepcr and FCM. 5) select THP-1 cells as model cells of human macrophages, and verify the expression of GDF15 to THP-1 cells and their polarization phase membrane molecules and cell causes by real-timepcr, ELISA and FCM. The effect of subsecretion and the selection of tgf- beta 1, which is most significantly affected by GDF15, further study the molecular mechanism of GDF15 regulation of tgf- beta 1 expression through the methods of double luciferase reporter gene system, real-timepcr, ELISA, Westernblot, transcription factor active chips, laser confocal, chip and RNA interference. The following results are as follows: 1) GDF15 directly induces the polarization of cd14+ monocytes to M2 like macrophages, which can inhibit the polarization of M1 type macrophages and induce the polarization of M2 like macrophages at the stage of macrophage polarization from cd14+ monocytes; 2) m2 like macrophages after GDF15 stimulation can induce Treg formation; 3) GDF15 can significantly induce CD14+ mononuclear cells and their origin. The expression of TGF- beta 1; 4) GDF15 can induce the polarization of mouse myelinated macrophages and their polarization phase to M2 like macrophages and inhibit the ability to stimulate the proliferation of T cells. 5) overexpression of GDF15 can promote the growth of the tumor in vivo, increase the ratio of M2 like macrophages and M2 like TAM infiltration in the abdominal cavity; 6) plasma GDF15 level, CD206+TAM immersion The ratio of moisture and the expression level of TGF- beta 1 of TAM, 22 positive correlation between three, and 7) GDF15 can induce THP-1 cells and their polarization stage to express TGF- beta 1; 8) GDF15 through K-Ras-JNK/ERK-c-Myc pathway to regulate the expression of TGF- beta 1. We first confirmed that GDF15 can induce macrophages to produce M2 like form and function; GDF15 inhibit macrophage polarization to M1 type. It leads to M2 like polarization; GDF15 significantly induces macrophage and its polarization stage to express the immunosuppressive factor TGF- beta 1; GDF15 in CT26 cell overexpression can improve its tumorigenicity. To clarify how GDF15 induces macrophages to express TGF- beta 1, we study the related mechanisms, thus providing a new research prescription for the occurrence of tumor immune tolerance. To provide new ideas and new targets for blocking tumor immune escape and improving tumor immunotherapy effect.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R735.35

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