大肠癌肿瘤微环境中肿瘤相关巨噬细胞通过下调microRNA-195-5p靶向调控MAGE的表达

发布时间：2018-06-01 01:49

本文选题：肿瘤相关巨噬细胞 + 肿瘤微环境　；参考：《苏州大学》2016年硕士论文

【摘要】：已有大量的研究证据证明在恶性肿瘤微环境(TME)中M2型巨噬细胞的含量表现为明显升高,在癌症组织中,肿瘤相关巨噬细胞被招募到肿瘤细胞分泌的细胞因子的肿瘤间质中来,从而促进肿瘤细胞的侵袭和转移,而肿瘤间质正是构成肿瘤微环境的主要组成成分。在这项研究中,我们侧重于研究TME中免疫细胞中的M2巨噬细胞在结肠肿瘤间质中作用于microRNA-195-5p的这一过程。首先研究microRNA-195-5p以及其靶基因MAGE的作用机理,先建立一个共培养体系使THP-1转化为M 2的巨噬细胞。方法是从培养的结肠癌细胞(LoVo细胞株)提取培养基上清液,用佛波酯(PMA,美国sigma公司产)和LoVo上清液一起刺激THP-1细胞转化为M2型巨噬细胞;第二步验证转化好的巨噬细胞表型,方法为:ELISA和流式细胞术测定特定的细胞因子表达;第三步:提取M2巨噬细胞中miR-195-5p用RT-PCR和免疫印迹证实了miR-195下调,同时它的靶蛋白MAGE升高,接下来,transwell侵袭试验表明,LoVo细胞及巨噬细胞共培养后上调和下调巨噬细胞内miR-195-5p,分别可以使LoVo细胞侵袭能力下降和上升。结肠癌是消化系统最常见的恶性肿瘤之一,近年来随着人们生活水平的提高和饮食结构的改变,其发病率呈逐年上升趋势。目前结肠癌的早期诊断仍然比较困难,多数患者诊断时己有远处转移。现今肿瘤微环境对肿瘤的影响成为新的研究热点,而肿瘤相关巨噬细胞(tumor associated macrophage,TAM)作为其中重要组成成分,在恶性肿瘤发生、发展及转移中的作用尤为突出。肿瘤相关巨噬细胞位于肿瘤间质,它可以通过产生细胞因子或直接调控基因及靶蛋白作用于肿瘤细胞,从而改变恶性肿瘤的生长、增殖、侵袭和转移等生物学特性。既往研究中发现肿瘤相关巨噬细胞与大多数癌症都密切相关,同时,它的浸润程度与这些肿瘤患者的预后呈负相关。本研究通过建立上述共培养体系并检测微小RNA-195-5p的变化,探讨肿瘤相关巨噬细胞对结肠癌生物学行为的影响。研究目的1.建立共培养模型,验证共培养体系中细胞成分为M2型巨噬细胞。2.M2型巨噬细胞的下行调控microRNA-195-5p,从而使MAGE表达量增加,进而影响结肠癌的生物学特性。
[Abstract]:There has been a lot of evidence that M2 macrophages are significantly increased in malignant tumor microenvironment (TME). In cancer tissues, tumor-associated macrophages are recruited into the tumor stroma of cytokines secreted by tumor cells. Thus, the invasion and metastasis of tumor cells are promoted, and tumor stroma is the main component of tumor microenvironment. In this study, we focused on the process by which M2 macrophages from immune cells in TME act on microRNA-195-5p in the stroma of colon tumors. Firstly, the mechanism of microRNA-195-5p and its target gene MAGE was studied. Firstly, a co-culture system was established to transform THP-1 into M 2 macrophages. The method was to extract the supernatant from the culture medium of colon cancer cell line LoVo, and to stimulate the transformation of THP-1 cells into M2 macrophages by using PMA and LoVo supernatants. The second step was to verify the phenotype of transformed macrophages. Methods miR-195-5p was extracted from M2 macrophages to determine the expression of specific cytokines by using a specific Elisa and flow cytometry. MiR-195 was down-regulated by RT-PCR and Western blotting, while its target protein MAGE was increased. Furthermore, transwell invasion assay showed that LoVo cells could up-regulate and down-regulate miR-195-5pafter co-culture of LoVo cells and macrophages, which decreased and increased the invasiveness of LoVo cells, respectively. Colon cancer is one of the most common malignant tumors in digestive system. In recent years, with the improvement of people's living standard and the change of dietary structure, the incidence of colon cancer is increasing year by year. At present, the early diagnosis of colon cancer is still difficult, most patients have distant metastasis. Nowadays, the influence of tumor microenvironment on tumor has become a new research hotspot. As an important component of tumor associated macrophage, tumor associated macrophage plays an important role in the occurrence, development and metastasis of malignant tumor. Tumor-associated macrophages are located in the stroma of tumor. They can act on tumor cells by producing cytokines or directly regulating genes and target proteins, thus changing the biological characteristics of malignant tumor such as growth, proliferation, invasion and metastasis. Previous studies have found that tumor-associated macrophages are closely associated with most cancers, and their degree of infiltration is negatively correlated with the prognosis of these patients. In this study, the effects of tumor associated macrophages on the biological behavior of colon cancer were investigated by establishing the above co-culture system and detecting the changes of small RNA-195-5p. Objective 1. A co-culture model was established to verify the down-regulation of microRNA-195-5p. the cell component of the co-culture system was M2 macrophage. 2. The microRNA-195-5p was regulated by microRNA-195-5p. thus the expression of MAGE was increased and the biological characteristics of colon cancer were affected.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.34

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