小鼠巨噬细胞与肿瘤相关巨噬细胞的比较蛋白质组学研究

发布时间：2018-04-13 00:15

本文选题：肿瘤微环境 + 肿瘤相关巨噬细胞　；参考：《中国科学院北京基因组研究所》2016年博士论文

【摘要】：肿瘤微环境由肿瘤细胞及其周围的免疫细胞、成纤维细胞、内皮细胞、血管、信号分子及胞外基质等组成。肿瘤细胞与其周围的微环境间存在着密切的联系。肿瘤细胞可以通过自分泌和旁分泌的方式释放多种功能分子,进而促进其自身发生发展及微环境的稳态平衡状态。巨噬细胞作为占实体肿瘤体积近50%的免疫细胞,在肿瘤微环境中发挥重要作用。肿瘤相关巨噬细胞(Tumor Associated Macrophages, TAMs),作为肿瘤炎性反应的主要调节者,已被广泛报道可以通过释放多种炎性细胞因子,蛋白酶及生长因子等调节肿瘤细胞的增殖,血管生成及抑制适应性免疫反应。根据近期报道,TAMs也可以通过释放一种纳米颗粒分子,exosome,调节受体细胞的功能。但迄今为止,关于TAMs及其分泌的exosome的蛋白质组成了解较少。在此,我们利用小鼠巨噬细胞系Ana-1并结合肿瘤培养基诱导的方法,得到TAMs细胞模型,对Ana-1和TAMs来源的全细胞,exosome及exosome-free蛋白组分进行定量蛋白质组学的比较分析。TAMs细胞全蛋白质组的分析结果提示肿瘤分泌的功能物质能够使得巨噬细胞的代谢水平发生显著改变,如氨基酸代谢,糖类代谢及脂肪酸代谢等。这一结果提示,在肿瘤微环境中,除了可以调节周围基质细胞的信号通路外,肿瘤细胞也可能通过改变基质细胞代谢水平的方式调节其功能。此外,全细胞蛋白质组学数据提示,在TAMs细胞中,多个糖皮质激素(GR)的上游及下游分子发生上调,通过实验发现,GR的入核水平在TAMs细胞中呈增加趋势,其下游的抗炎症细胞因子,如IL-10及IL-1RA的mRNA表达增加。这个结果提示,GR信号通路的激活可能是本研究中TAMs模型表现出抗炎症表型的潜在原因。Ana-1及TAMs来源的exosome及exosome-free的蛋白质组学研究提示,主要通过exosome形式分泌的蛋白质富集于RNA加工及蛋白酶解相关的通路,而与代谢稳态相关的蛋白质主要通过exosome-free的形式进行分泌。通过比较小鼠巨噬细胞来源的exosome蛋白质组及其他已被报道的三种细胞来源的exosome蛋白质组,发现巨噬细胞来源的exosome具有细胞来源特异性,主要集中在RNA加工及核糖体生物合成等过程。而根据蛋白质组学数据,细胞从Ana-1变为TAMs的过程,显著改变了其分泌的exosome中20S蛋白酶体亚基及核糖体蛋白的丰度。20S蛋白酶体活性检测结果表明,TAMs来源的exosome具有更强的蛋白酶解活性。此外,Ana-1及TAMs来源的exosome具有不同的RNA组成及丰度,这样的现象可能与两种exosome中包裹了不同含量的RNA结合蛋白有关。这些结果提示,通过TAMs田胞释放的exosome可能会通过降解错误折叠蛋白或传递特异RNA到受体肿瘤细胞的形式调节肿瘤细胞的发生发展过程。总而言之,本研究通过对Ana-1和TAMs的全细胞蛋白组、exosome及exosome-free的蛋白质组的全面分析及相关功能验证,揭示了TAMs及其分泌的exosome的分子功能特征,为了解肿瘤微环境中TAMs,尤其是TAMs来源的exosome的复杂功能提供新的视角。
[Abstract]:The tumor microenvironment by tumor cells and peripheral immune cells, fibroblasts, endothelial cells, blood vessels, signaling molecules and extracellular matrix components. Microenvironment of tumor cells and their surrounding are closely linked. Tumor cells through autocrine and paracrine way to release a variety of functional molecules, thereby promoting homeostasis the state of its own development and micro environment. As for macrophage immune cells in solid tumor volume of nearly 50%, play an important role in the tumor microenvironment. Tumor associated macrophages (Tumor Associated, Macrophages, TAMs), as a major regulator of tumor inflammatory response, has been widely reported by inflammatory cytokines, protease and growth factor regulating the proliferation of tumor cells, and inhibit the generation of adaptive immune response vessels. According to recent reports, TAMs can also be through the release of A nanoparticle molecule, exosome, regulation of receptor cell function. But so far, about TAMs and secreted exosome protein composition are poorly understood. Here we use the mouse macrophage cell line Ana-1 and tumor cell culture medium for inducing method, TAMs cell model, whole cell of Ana-1 and TAMs sources, comparative analysis exosome and exosome-free protein were quantitative proteomic analysis of.TAMs cell proteome results suggest that functional substances secreted by tumor can make the metabolism of macrophages changed significantly, such as amino acid metabolism, carbohydrate metabolism and fatty acid metabolism. The results suggest that in the tumor microenvironment, in addition to regulating signal pathway the surrounding stromal cells, tumor cells may also regulate its function by changing the stromal cells metabolism. In addition, the whole cell proteome The data suggest that in TAMs cells, a plurality of Glucocorticoid (GR) in the upstream and downstream molecules were up-regulated, found through experiments, GR into the nucleus level was increased in TAMs cells, the anti-inflammatory cytokine downstream, such as IL-10 and IL-1RA and increased the expression of mRNA. The results suggest that the activation of GR signaling pathway may be the TAMs model in this study showed exosome and exosome-free anti-inflammatory phenotype and potential reasons for.Ana-1 and TAMs derived proteomics research suggests, mainly through the form of exosome secreted proteins enriched in RNA processing and protein enzyme related pathways and related metabolic homeostasis mainly through protein the form of exosome-free exosome exosome protein secretion. Through the comparative analysis of the proteome of mouse macrophages and other sources have been reported to be three kinds of cells, macrophage source exos Ome has specific cell sources, mainly concentrated in the process of RNA processing and ribosome biogenesis. According to proteomics data, cells from Ana-1 to TAMs, significantly changed the abundance of.20S protein enzyme activity detection results of 20S PROTEASE SECRETING exosome ribosomal subunits and ribosomal protein showed that the source of TAMs exosome has stronger proteolytic activity. In addition, Ana-1 and TAMs exosome from RNA with different composition and abundance, this phenomenon may be related to the two exosome package with different content of RNA binding proteins. These results suggest that TAMs cells released by Tian exosome may through the degradation of misfolded proteins or the development process of transfer form to tumor cell specific RNA receptor regulates tumor cell. In short, through the whole cell protein of Ana-1 and TAMs in this study, exosome and exosome The comprehensive analysis of -free proteome and related functional verification reveal the molecular function characteristics of TAMs and its secreted exosome, and provide a new perspective for understanding the complex functions of TAMs in the tumor microenvironment, especially the TAMs derived exosome.

【学位授予单位】：中国科学院北京基因组研究所
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R73-3

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