GDF15影响巨噬细胞极化及其在介导结肠癌免疫逃逸中的作用研究

发布时间：2018-05-18 03:16

本文选题：GDF15 + 巨噬细胞　；参考：《第四军医大学》2016年博士论文

【摘要】：全球范围内恶性肿瘤的发病率仍将处于不断增长的趋势。近年来,肿瘤免疫治疗因其良好的临床疗效,得到越来越多的重视和认可。然而,肿瘤免疫逃逸严重限制了肿瘤免疫治疗的效果。研究表明,肿瘤微环境下免疫细胞的功能失调在肿瘤免疫逃逸过程中发挥重要作用。巨噬细胞是固有免疫系统中重要的细胞组成部分,在抗感染、提呈抗原启动免疫应答、抗肿瘤及免疫调节过程中发挥重要作用。在不同微环境的影响下,巨噬细胞分化为不同的表型并表现出不同的功能,即极化现象。目前将巨噬细胞主要分为两大类,即以分泌促炎因子为主的经典活化M1型巨噬细胞和以发挥消炎反应、促进组织修复功能为主的替代性活化M2型巨噬细胞。其中,M1型巨噬细胞可诱导Th1反应的发生,促进炎性因子(如IL-12等)介导的Th1细胞应答,杀伤胞内寄生菌和肿瘤细胞;M2型巨噬细胞通过支持Th2细胞相关的效应功能,在抑制机体免疫系统杀伤肿瘤细胞的过程中发挥作用。大量的临床和实验证据表明,在大多数肿瘤中,肿瘤组织浸润的巨噬细胞,即肿瘤相关巨噬细胞,普遍具有m2样表型,可分泌多种生长因子促进肿瘤血管生成,分泌多种参与基质降解的酶类促进肿瘤的侵袭和转移,诱导treg细胞分化进而抑制cd8+t细胞的细胞毒性,抑制ctl细胞和nk细胞的增殖和杀伤功能、抑制dc细胞的活化、成熟和抗原提呈功能,从而参与肿瘤免疫逃逸,促进肿瘤的恶性发展。gdf15首次从人单核细胞系u937的巨噬细胞活化相关基因cdna文库中鉴定并表达,是tgf-β超家族的成员,参与了多种生理和病理过程。gdf15的表达失调还与多种肿瘤的发生发展密切相关,并直接影响患者的预后和生存质量。虽然gdf15作为肿瘤源性因子在肿瘤的发生发展过程中的作用已有较多关注,但时至今日,关于gdf15对免疫细胞作用的明确报道依然廖廖无几。我们前期研究了gdf15对dc及treg细胞分化成熟和功能的影响,首次证实gdf15不仅在dc的成熟和功能发挥过程中发挥显著的抑制效应,还可以通过上调foxp3表达,促进naivecd4+t细胞向treg分化成熟。那么,gdf15是否可影响巨噬细胞的极化?其诱导巨噬细胞发挥免疫抑制功能的机制又是什么?为阐明上述问题,我们进行了以下研究工作:1)首先从人外周血单个核细胞中分离得到cd14+单核细胞,进一步诱导获得巨噬细胞。通过real-timepcr、elisa和fcm等方法研究gdf15对cd14+单核细胞及其来源巨噬细胞极化阶段膜分子表达、细胞因子分泌、吞噬能力及诱导na?vecd4+t细胞表达foxp3能力的影响;2)利用慢病毒感染构建gdf15过表达的小鼠结肠癌ct26稳转细胞系;3)通过real-timepcr、fcm和mlr等方法研究gdf15对鼠髓源性巨噬细胞及其极化阶段膜分子表达、细胞因子分泌、no分泌及刺激t细胞增殖能力的影响;4)构建荷瘤小鼠,通过elisa、real-timepcr和fcm等方法研究gdf15的体内促瘤作用及对tam的影响;5)选取thp-1细胞作为人巨噬细胞的模式细胞,通过real-timepcr、elisa和fcm等方法验证gdf15对thp-1细胞及其极化阶段膜分子表达和细胞因子分泌的影响,并选择其中受gdf15影响最显著的tgf-β1,进一步通过双荧光素酶报告基因系统、real-timepcr、elisa、westernblot、转录因子活性芯片、激光共聚焦、chip和rna干涉等方法研究gdf15调控tgf-β1表达的分子机制。通过以上研究工作,我们得到了如下结果:1)gdf15直接诱导cd14+单核细胞向m2样巨噬细胞极化,在cd14+单核细胞来源巨噬细胞极化阶段可抑制m1型巨噬细胞极化,诱导m2样巨噬细胞极化;2)gdf15刺激后的m2样巨噬细胞能够诱导Treg形成;3)GDF15可显著诱导CD14+单核细胞及其来源巨噬细胞在极化阶段表达TGF-β1;4)GDF15可诱导鼠髓源性巨噬细胞及其极化阶段向M2样巨噬细胞极化,并抑制其刺激T细胞增殖的能力;5)过表达GDF15可促进肿瘤体内生长,增加腹腔M2样巨噬细胞比率及M2样TAM浸润;6)血浆GDF15水平、CD206+TAM浸润比率及TAM的TGF-β1表达水平,三者之间成两两正相关;7)GDF15可诱导THP-1细胞及其极化阶段表达TGF-β1;8)GDF15通过K-Ras-JNK/ERK-c-Myc通路调控TGF-β1表达。我们首次证实GDF15可诱导巨噬细胞产生M2样表型和功能;GDF15抑制巨噬细胞向M1型极化,诱导其向M2样极化;GDF15显著诱导巨噬细胞及其极化阶段表达免疫抑制性因子TGF-β1;GDF15在CT26细胞过表达可提高其成瘤活性。为阐明GDF15如何诱导巨噬细胞表达TGF-β1,我们对相关机制进行研究,从而为肿瘤免疫耐受的发生提供新的研究方向,为阻断肿瘤免疫逃逸、提高肿瘤免疫治疗效果提供新思路和新靶点。
[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.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.35

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