Rab6对吞噬活性的调控及巨噬细胞在肿瘤转移中的作用

发布时间：2018-03-07 05:26

本文选题：巨噬细胞　切入点：Rab6　出处：《浙江大学》2016年博士论文　论文类型：学位论文

【摘要】：巨噬细胞是参与体内组织防御和稳态维持的一群重要的免疫细胞,一方面它可以利用吞噬作用来杀死和清除病原体、细胞碎片及损伤组织,另一方面它可以激发获得性免疫反应从而发挥进一步的功能。巨噬细胞具有异质性,在不同环境信号刺激下呈现不同表型和功能。目前根据参与的免疫应答反应将巨噬细胞分为经典活化的巨噬细胞(M1型)和替代性活化的巨噬细胞(M2型)。M1型巨噬细胞主要参与1型辅助T细胞(Th1)抗病原的免疫应答反应,可有效杀死病原菌和肿瘤细胞；而M2巨噬细胞抑制免疫反应和获得性免疫应答,参与血管生成、组织重排和修复等过程。肿瘤组织中的巨噬细胞[肿瘤相关巨噬细胞(tumor associated macrophages, TAM)]多呈现M2表型,它们并非发挥抗肿瘤作用,而是可以促进肿瘤发生、增值、入侵和转移等过程,并且与肿瘤组织中血管生成有密切关系。因此,研究M1型巨噬细胞在免疫中的作用以及M2巨噬细胞在肿瘤发展中的作用对理解异质的巨噬细胞的不同功能具有积极的意义。M1型巨噬细胞作为专业的吞噬细胞,在抗菌感染过程中发挥着重要的作用。在组织被病原感染之后,最先响应的巨噬细胞通常呈现出炎性表型并分泌大量的促炎分子。巨噬细胞的吞噬作用是动物进化中较保守的细胞生理活动,主要行使防御病原菌侵染宿主和起始获得性免疫应答的功能。Rab蛋白是细胞质中囊泡运输的分子开关,目前发现多种Rab蛋白参与吞噬过程。本实验室前期研究表明,在无脊椎动物对虾和果蝇中,Rab6蛋白通过直接与肌动蛋白互作来调控细胞吞噬作用,从而抵抗病毒的感染。但是,Rab6蛋白在哺乳动物吞噬作用中的功能尚不清楚。本论文以小鼠巨噬细胞RAW 264.7为研究对象,探索了小G蛋白Rab6在哺乳动物吞噬过程中的作用。研究结果显示,Rab6蛋白不直接影响巨噬细胞对金黄色葡萄球菌的摄取,而是参与胞内吞噬小体的成熟过程。Rab6蛋白与其效应蛋白BICD1相互作用,调控巨噬细胞内吞噬小体成熟的中后期,这一作用机制与无脊椎动物不同。Rab6蛋白参与的吞噬作用对吞入的金黄色葡萄球菌在胞内的存活也有消极的影响。通过非活性和活性状态的转变以及与驱动蛋白复合物的相互作用,Rab6蛋白可以在时空上控制马达蛋白介导的运输过程和细胞内结构的融合过程。因此,本论文揭示了小G蛋白Rab6在调控哺乳动物巨噬细胞抗菌免疫中的重要作用。另一方面,我们研究了M2型巨噬细胞在肿瘤转移中的作用。在肿瘤组织中,除了肿瘤细胞之外还存在其他类型的细胞,包括成纤维细胞、内皮细胞和免疫细胞,这些细胞共同组成肿瘤微环境。肿瘤组织中最重要的免疫细胞是巨噬细胞,且多呈现M2表型。本论文中免疫组织化学染色结果显示,胃癌组织周围聚集大量的M2型巨噬细胞,说明肿瘤细胞将巨噬细胞招募至组织中,并诱导其分化为M2表型。TAM通过分泌细胞因子参与肿瘤发展的调控,但是目前关于巨噬细胞分泌蛋白在肿瘤发生中的作用研究仍然较少。本文的细胞试验和动物试验结果显示,M2型巨噬细胞大量分泌的几丁质酶3样蛋白1 (CHI3L1)具有促肿瘤转移的活性。通过GST下拉试验,我们寻找到与CHI3L1结合的肿瘤细胞膜上的受体——白介素13受体a2 (IL-13Rα2);细胞免疫荧光和免疫共沉淀试验证明了两者之间的直接互作。由此可知,CHI3L1蛋白通过与肿瘤细胞膜上的IL-13Rα2受体相互作用,进而促进肿瘤细胞的迁移、粘附和入侵,以达到促肿瘤转移的目的。研究结果显示,CHI3L1蛋白处理肿瘤细胞后,IL-13Rα2受体的激活触发细胞内丝裂原活化蛋白激酶(MAPK)信号通路,引起细胞外调节蛋白激酶1和2(ERK1/2)和c-Jun氨基末端激酶(JNK)蛋白磷酸化水平的升高,进而促进激活蛋白1(AP-1)家族转录因子在细胞核内的聚集。AP-1家族转录因子可以结合在基质金属蛋白酶(MMP)基因的启动子区域,从而促进MMP家族基因的转录。肿瘤细胞分泌的MMP可以有效降解基底膜和周围的胞外基质,使纤连蛋白暴露于肿瘤细胞之间,从而促进肿瘤细胞的粘附和转移。与此同时,动物试验结果表明CHI3L1蛋白具有促肿瘤转移的功能。因此,巨噬细胞中的CHI3L1蛋白可以作为肿瘤治疗的新靶点。我们采用免疫共沉淀与Western blot相结合的方法来检测入血清样品中CHI3L1蛋白的含量,结果显示,乳腺癌和胃癌病人血清中CHI3L1蛋白的含量较高,而正常人血清中CHI3L1蛋白检测不到。此前有研究报道,在乳腺癌和肺癌中,癌症病人血清中HER2和C反应蛋白的含量明显高于健康人,并可作为肿瘤诊断的标志物。由此可见,血清中的CHI3L1蛋白有望成为诊断乳腺癌和胃癌的标志蛋白。在了解了M2型巨噬细胞分泌的CHI3L1蛋白促肿瘤转移的作用机制后,我们以CHI3L1基因作为靶点,探索对虾microRNA (miRNA)在跨物种调控人类肿瘤转移中的作用。为了研究对虾miRNA能否跨物种调控人肿瘤的转移,我们在M2型巨噬细胞内过表达对虾miRNA,进而检测肿瘤细胞转移能力的变化。我们利用生物信息学软件预测了三条可能与人CHI3L1 mRNA 3'UTR(3'非编码区)相互作用的对虾特有的miRNA——mja-miR-35、mja-miR-36、mja-miR-37。细胞转染以及靶基因验证试验表明,对虾mja-miR-35可以直接作用于CHI3L1 mRNA 3'UTR,敲低巨噬细胞内CHI3L1基因的表达,并减少分泌到胞外的CHI3L1蛋白的含量,从而影响巨噬细胞对肿瘤细胞转移的促进作用。但是mja-miR-36和mja-miR-37并不能有效敲低CHI3L1基因的表达量。凝胶迁移阻滞试验显示,人AG02蛋白与这三条miRNA均能结合,说明AG02蛋白与miRNA的结合没有序列和物种选择性。我们推测mja-miR-36、mja-miR-37与AG02蛋白的结合可能改变了AG02蛋白的构象,从而抑制了AG02蛋白介导的靶基因降解过程。对虾作为重要的水产品之一,由于感染对虾白斑综合症病毒(WSSV)引起的对虾大量死亡是对虾养殖的难题。近几年研究发现,siRNA和miRNA参与对虾的抗病毒免疫。我们通过研究发现,对虾mja-miR-35可以作用于WSSV病毒基因,从而抑制病毒在对虾体内的复制及感染,促进对虾的抗病毒免疫。这些结果说明,对虾mja-miR-35既能调控对虾抗病毒免疫,又能跨物种调控人类肿瘤细胞的转移。本论文围绕巨噬细胞在抗菌免疫以及促进肿瘤转移中的功能展开研究。首先通过研究小G蛋白Rab6在哺乳动物M1型巨噬细胞吞噬过程中的作用,我们发现Rab6蛋白能调控吞噬小体的成熟过程。其次,我们阐明了肿瘤细胞可以招募M2型巨噬细胞以及M2型巨噬细胞大量分泌的CHI3L1蛋白进一步促进肿瘤转移的作用机制。因此,本文的研究结果对理解巨噬细胞在抗菌免疫以及肿瘤转移中的功能具有重要意义。最后,我们发现一条对虾miRNA——mja-miR-35,该miRNA既能调控对虾抗病毒免疫,又能跨物种调控人类肿瘤细胞的转移。
[Abstract]:Macrophages are the immune cells involved in tissue defense and homeostasis maintained a group of important, on the one hand it can be used to kill and eliminate pathogens phagocytosis, cell debris and tissue damage, on the other hand, it can stimulate the acquired immune response and play a further function. Macrophages are heterogeneous and exhibit different phenotype and function in different environment according to the current signal stimulation. The immune response in macrophages into classically activated macrophages (M1) and alternatively activated macrophages (M2).M1 macrophages mainly involved in type 1 T helper cell (Th1) antiviral immune response the reaction, which can effectively kill pathogens and tumor cells; M2 inhibition of macrophage immune response and acquired immune response, angiogenesis, tissue repair and rearrangement process. In tumor tissue macrophages [tumor Related (tumor associated macrophages, macrophage TAM)] showed M2 phenotype, they do not exert anti-tumor effect, but added can promote tumorigenesis, invasion and metastasis process, and vascular tumor formation are closely related. Therefore, different functions of macrophages induced inhibition of M1 macrophages in immunity and M2 macrophages in tumor development in understanding the heterogeneity of the significance of.M1 positive macrophages as professional phagocytes, antibacterial infection plays an important role in the process. After the organization was the first response to pathogen infection, macrophages are usually inflammatory phenotype and secretion of proinflammatory molecules. The phagocytosis of macrophage is a relatively conserved cellular physiological activity in animal evolution, the main pathogen infection and host defense exercise initiation acquired immune response. .Rab protein is vesicular transport in the cytoplasm of the molecular switch, found that many Rab proteins involved in the phagocytic process. Our previous study showed that in invertebrates, shrimp and Drosophila, Rab6 protein through direct interaction with actin to regulate cell phagocytosis, and resistance to virus infection. However, the function of Rab6 protein in mammalian phagocytosis in is not clear. The mouse macrophage RAW 264.7 as the research object, to explore the small G protein Rab6 in mammalian phagocytic process. The results showed that the Rab6 protein does not directly affect the uptake of macrophages on Staphylococcus aureus, but interactions involved in intracellular phagosome maturation process of.Rab6 protein and its effect BICD1 protein, regulation of macrophage phagosome maturation in the late, this mechanism with different.Rab6 proteins in invertebrates The phagocytosis of ingestion of Staphylococcus aureus in intracellular survival can also have a negative impact. Through the transformation of non active and active state and interaction with kinesin complexes, Rab6 protein can control the transport process of motor proteins and cell fusion mediated within the structure of the process in time and space. Therefore, the this paper reveals the small G protein Rab6 in regulating mammalian macrophages play an important role in antibacterial immunity. On the other hand, we study the M2 type macrophage function in tumor metastasis. In tumor tissues, in addition to the tumor cells also exist in other cell types, including fibroblasts, endothelial cells and immune cells, these the cell is composed of the tumor microenvironment in tumor tissue. The most important immune cells are macrophages, and showed M2 phenotype. The immunohistochemical staining results showed that the gastric cancer group A large number of fabric gathered around M2 macrophages, tumor cells will explain macrophage recruitment to the tissue, and induce the differentiation phenotype of M2.TAM regulates secretion of cytokines involved in tumor development through, but the current research on the role of macrophage secretory protein in tumorigenesis is still small. The cell and animal experiments results in this paper show that M2 a large number of macrophages secrete 3 chitinase like protein 1 (CHI3L1) can promote tumor metastasis activity. Through the GST drop-down test, we find the tumor cell membrane and CHI3L1 binding on the receptor - interleukin 13 receptor A2 (IL-13R alpha 2); test between direct immunofluorescence and immune interaction co precipitation. Therefore, CHI3L1 protein with tumor cell membrane IL-13R alpha 2 receptor interaction, thereby promoting tumor cell migration, adhesion and invasion, in order to To promote tumor metastasis. The results of the study showed that CHI3L1 protein in tumor cells after activation of IL-13R alpha 2 receptors trigger intracellular mitogen activated protein kinase (MAPK) signaling pathway induced by extracellular regulated protein kinase 1 and 2 (ERK1/2) and c-Jun N-terminal kinase (JNK) phosphorylation level increased. And then promote the activator protein 1 (AP-1) family of transcription factors in the nucleus of the aggregation of.AP-1 family transcription factors bind to matrix metalloproteinase (MMP) gene promoter, so as to promote the transcription of MMP gene family. The extracellular matrix secreted by tumor cells, MMP can effectively degrade the basement membrane and the surrounding, the fibronectin exposure to tumor cells, so as to promote the adhesion and metastasis of tumor cells. At the same time, animal experiment results show that CHI3L1 protein can promote tumor metastasis. Therefore, macrophage CHI3L1 in eggs A new target white can be used as tumor therapy. We use the content of CO immunoprecipitation method combined with Western blot to detect CHI3L1 protein in serum samples showed higher content of serum CHI3L1 protein in patients with breast cancer and gastric cancer, and the detection of CHI3L1 protein in normal human serum. Previous studies have reported that in breast cancer and lung cancer, cancer, HER2 and C content in serum of patients with C-reactive protein was significantly higher than that of healthy people, and can be used as a marker of tumor diagnosis. Thus, the serum CHI3L1 protein is expected to become a diagnosis of breast cancer and gastric cancer marker protein. In the understanding of the M2 type macrophage CHI3L1 protein promoting mechanism tumor metastasis, we used CHI3L1 as a target gene, microRNA (miRNA) to explore the shrimp in cross species regulation of human tumor metastasis. In order to study whether miRNA can cross species of shrimp Regulation of human tumor metastasis, we overexpressed miRNA in shrimp M2 macrophages, and detecting the changes of metastatic ability of tumor cells. We use bioinformatics software predicted three possible CHI3L1 mRNA 3'UTR (3'and non encoding regions) the interaction of shrimp specific miRNA - mja-miR-35, mja-miR-36, showed that mja-miR-37. cell transfection and the target gene test, shrimp mja-miR-35 can have a direct effect on the CHI3L1 mRNA 3'UTR on CHI3L1 gene expression in macrophages is low, and reduce the content of extracellular CHI3L1 protein, thus affecting macrophages on tumor cell metastasis promoting effect. But mja-miR-36 and mja-miR-37 can not effectively at low expression of CHI3L1 gene of the gel. Transfer block test showed that the human AG02 protein and the three miRNA can be combined with AG02 and miRNA protein showed no sequence and species selection Optional. We speculate that mja-miR-36, the combination of mja-miR-37 and AG02 protein may alter the conformation of AG02 protein, thus inhibiting the degradation of target genes mediated by AG02 protein. The shrimp is one of the important aquatic products, due to the infection of white spot syndrome virus (WSSV) caused by the death of a large number of shrimp shrimp farming is a difficult problem in recent. Years of research found that siRNA and miRNA are involved in antiviral immunity of shrimp. We found that the shrimp mja-miR-35 can act on the WSSV virus gene, thereby inhibiting virus replication and infection in shrimp body, promote the antiviral immunity of shrimp. These results suggest that the shrimp mja-miR-35 can both regulate the antiviral immunity of shrimp, and cross species transfer regulation of human tumor cells. This paper focuses on the research on macrophage antibacterial immunity and promote tumor metastasis function. First through the study of small G protein Rab 6 macrophages in mammalian type M1 in the process of phagocytosis, we found that Rab6 protein can regulate phagosome maturation process. Secondly, we clarify the tumor cells can recruit M2 macrophages and M2 macrophages to secrete CHI3L1 protein to further promote the mechanism of tumor metastasis. Therefore, the results of this study have important understanding of macrophage in the antibacterial immunity and function in tumor metastasis. Finally, we found a miRNA - mja-miR-35, the miRNA can control the antiviral immunity of shrimp, and cross species transfer regulation of human tumor cells.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R73-37

【相似文献】