异甘草酸镁调节胃癌炎性微环境的抑癌机制研究

发布时间：2019-06-09 16:14

【摘要】：肿瘤的发生、发展和侵袭转移与炎症密切相关,免疫细胞和炎症因子几乎影响到肿瘤进展的每一方面。肿瘤相关性炎症已被列入肿瘤的第七种特征,肿瘤相关炎症能够通过促进血管新生、促进肿瘤转移、抑制抗肿瘤免疫应答及改变肿瘤细胞对化疗药的敏感性等方面促进肿瘤的发生和进展,而这种作用主要依赖肿瘤微环境(TME)实现。因此调节其炎性微环境可能会翻开肿瘤治疗策略的新一页。胃癌作为世界第五大常见的恶性肿瘤,发现晚,治疗难,考虑其与幽门螺杆菌(Hp)感染和慢性胃炎关联甚密,我们试图找到通过调节其炎性微环境控制肿瘤的方法。临床常用药异甘草酸镁(MgIG)被发现的抗癌疗效为我们提供了思路。异甘草酸镁是甘草酸的第四代制剂,具有抗炎保肝、抗病毒、抗氧化、抗过敏、调节免疫和抗癌疗效。课题组也在前期工作中通过动物实验,验证了异甘草酸镁对胃癌的干预作用,在此基础上,我们对小鼠肿瘤组织中炎性细胞因子表达情况和炎性细胞密度进行了检测,在对此结果进行分析讨论的基础上,进一步选取巨噬细胞作为研究对象,体外模拟肿瘤的炎性微环境,观察异甘草酸镁对微环境中巨噬细胞极化的影响,以此探讨异甘草酸镁是否通过调节胃癌炎性微环境从而干预胃癌。第一部分异甘草酸镁对胃癌小鼠微环境中炎症细胞因子表达和炎性细胞的影响研究背景:已知异甘草酸镁处理皮下荷瘤的小鼠后小鼠瘤重减轻,瘤体积缩小,生存时间延长。同时已知异甘草酸镁对炎症因子和炎性细胞的调节作用,以及胃癌的炎性微环境对胃癌进展的不可忽视的作用。目的:检测胃癌小鼠肿瘤组织中免疫细胞和炎症因子,发现异甘草酸镁是否通过对胃癌炎性微环境的调节来抑制胃癌的发展。方法:首先我们选择了一款能检测96个与炎症和自身免疫相关的分子表达水平的转录组芯片来检测异甘草酸镁对小鼠胃癌移植瘤组织内这些重要的细胞因子的表达水平的影响,主要为趋化因子家族及受体、白介素家族及受体以及NF κB通路关键分子,并用qPCR对芯片结果进行了验证。然后我们用免疫组化对肿瘤微环境中的关键炎性细胞进行染色。结果:异甘草酸镁下调了小鼠胃癌组织中趋化因子及其受体CCL3、CCL5、CCL8、CCL11、CCL13、CCL19、CCL21、CXCL1、CXCL2、CXCL8、CCR1、CCR3、CCR4、CCR7、CXCR1、CXCR2的表达;下调了小鼠胃癌组织中TLRs、MyD88、Tollip、NF-κb的表达;下调了小鼠胃癌组织中il1a、il1b、il1r1、il1rap、il1rn、il5、il6、il6r、il10、il10rb、il15、il17a、il18、il22、il23a、il23r的表达,上调了il2、il12的表达;降低了小鼠胃癌组织中treg细胞密度和活性,增大了胃癌组织中cd8+t细胞密度,增大了胃癌组织中巨噬细胞的密度(p0.05)。结论:异甘草酸镁可能通过调节小鼠胃癌组织中趋化因子及其受体、白介素家族及受体以及nf κb通路关键分子的表达和treg细胞、cd8+t细胞的密度(和活性)而抑制胃癌的发展。第二部分异甘草酸镁对胃癌微环境中巨噬细胞极化的诱导作用研究背景:第一部分我们发现CD68+的巨噬细胞密度在异甘草酸镁用药组增大,而现在普遍认为巨噬细胞密度越高,预后越差(胰腺癌中除外),因此异甘草酸镁用药组巨噬细胞密度增加但小鼠生存期延长似乎与以往文献不符。但是巨噬细胞是一个异质的细胞群,具有高度的可塑性,在不同的环境下可分化为M1型(经典激活)和M2型(选择性激活)巨噬细胞,而这两种巨噬细胞的作用更是云泥之别,巨噬细胞在肿瘤微环境的作用下会分化为M2型。目的:阐明异甘草酸镁明显延长荷瘤小鼠生存期的机制:是否是通过诱导肿瘤微环境中巨噬细胞向M1分化实现的。方法:采用条件培养基模拟胃癌的微环境,先用不同浓度的异甘草酸镁溶液处理小鼠胃癌MFC细胞,然后收集上清,加到培养的小鼠巨噬细胞RAW264.7中,观察RAW264.7的极化情况,再分别收集RAW264.7细胞和上清,细胞提取蛋白western blot法检测iNOS、Arg1、IL12表达水平,上清ELISA法检测IL6、IL10、IL12、TGF-β1、TNF、VEGF-A等与肿瘤免疫密切相关因子,从而分析巨噬细胞极化情况。结果:胃癌MFC细胞可以诱导小鼠巨噬细胞RAW264.7分化为M2型(高表达Arg1,低表达iNOS、IL12),而在培养基中加入异甘草酸镁培养的胃癌MFC细胞上清,则诱导小鼠巨噬细胞RAW264.7分化为M1型(高表达iNOS、IL12,低表达Arg1);而未处理的MFC细胞上清培养RAW264.7后,上清中的IL6、IL10、TGF-β、TNF-α、VEGF-A,较DMEM完全培养基培养的RAW264.7上清中浓度显著升高,而IL12明显要低。在MFC培养上清中加入不同浓度异甘草酸镁后获得的条件培养基,再去培养RAW264.7,其IL6、IL10、TGF-β、TNF-α、VEGF-A浓度显著降低,IL12浓度升高。结论:异甘草酸镁可以通过调节肿瘤细胞的分泌间接诱导,和直接调控胃癌微环境中肿瘤相关巨噬细胞向M1型巨噬细胞复极化,这极有可能是异甘草酸镁干预胃癌发展的作用方式。
[Abstract]:The occurrence, development and invasion and metastasis of the tumor are closely related to the inflammation, and the immune cells and the inflammatory factors have little effect on every aspect of the progress of the tumor. The tumor-related inflammation has been included in the seventh characteristic of the tumor, and the tumor-related inflammation can promote the occurrence and progression of the tumor by promoting angiogenesis, promoting the tumor metastasis, inhibiting the anti-tumor immune response and changing the sensitivity of the tumor cell to the chemotherapeutic drug, This effect is mainly dependent on the tumor microenvironment (TME). Therefore, adjusting the inflammatory microenvironment of the tumor may turn a new page of the tumor treatment strategy. Gastric cancer, as the fifth most common malignant tumor in the world, has been found to be late and difficult to treat, considering that it is closely associated with H. pylori (Hp) infection and chronic gastritis, and we try to find a way to control the tumor by adjusting its inflammatory microenvironment. The anti-cancer curative effect of magnesium (MgIG), which is commonly used in clinical application, is a thought. Magnesium isoglycyrrhizinate is the fourth generation of glycyrrhizic acid, and has the effects of resisting inflammation, protecting liver, resisting virus, resisting oxidation, resisting allergy, regulating immunity and resisting cancer. The effect of magnesium isoglycyrrhizin on gastric cancer was verified by animal experiment in the earlier stage, and the expression of inflammatory cytokines and the density of inflammatory cells in the tumor tissues of mice were detected, and the results were analyzed and discussed. The effect of magnesium isoglycyrrhizin on the polarization of the macrophage in the microenvironment was studied by further selecting the macrophage as the research object and simulating the inflammatory microenvironment of the tumor in vitro. The influence of the first part of magnesium isoglycyrrhizin on the expression of inflammatory cytokines and inflammatory cells in the micro-environment of gastric cancer mice was studied. It is also known that the effect of magnesium isoglycyrrhizin on the inflammatory factors and inflammatory cells, as well as the non-negligible effect of the inflammatory microenvironment of gastric cancer on the progress of gastric cancer. Objective: To detect the immune cells and inflammatory factors in the gastric cancer mouse tumor tissue, and to find out whether the magnesium isoglycyrrhizin can inhibit the development of the gastric cancer by regulating the inflammatory microenvironment of the gastric cancer. Methods: First, we selected a transcriptome chip capable of detecting the expression level of 96 molecules related to inflammation and autoimmunity to detect the effect of magnesium isoglycyrrhizin on the expression level of these important cytokines in the tissue of mice with gastric cancer, mainly the chemokine family and the receptor. The key molecules of the interleukins family and the receptor and the NF-B pathway were tested and the results of the chip were verified by qPCR. The key inflammatory cells in the tumor microenvironment were then stained with immunohistochemistry. Results: The expression of the chemokine and its receptors, CCL3, CCL5, CCL8, CCL11, CCL13, CCL8, CCR1, CCR3, CCR4, CCR7, CXCR1, and CXCR2 in the gastric cancer tissues of the mice was reduced by magnesium isoglycyrrhizin, and the expression of the TLRs, MyD88, Tollip and NF-5b in the gastric cancer tissues of the mice was down-regulated. the expression of il1a, il1b, il1r1, il1rap, il1rn, il5, il6, il6r, il10, il10rb, il15, il17a, il18, il22, il23a and il23r in the gastric cancer tissues of the mouse is reduced, the expression of the il2 and il12 is upregulated, the density and the activity of treg cells in the gastric cancer tissue of the mouse are reduced, the density of the cd8 + t cells in the gastric cancer tissue is increased, The density of macrophages in gastric cancer was increased (p0.05). Conclusion: Magnesium isoglycyrrhizin may inhibit the development of gastric cancer by regulating the expression of the chemokine and its receptor, the interleukin-family and the receptor, and the expression of the key molecules of the nf-b pathway and the density (and activity) of the treg cells and cd8 + t cells in the gastric cancer tissues of the mice. The second part of the study on the effect of magnesium isoglycyrrhizin on the polarization of macrophages in the microenvironment of gastric cancer: The first part found that the density of the macrophages of CD68 + increased in the group of magnesium isoglycyrrhizin, and it is generally accepted that the higher the density of the macrophages, the worse the prognosis (except in the case of pancreatic cancer), Therefore, the increased macrophage density in the magnesium isoglycyrrhizin group, but the prolongation of the survival of the mice appeared to be inconsistent with the previous literature. but the macrophages are a heterogeneous population of cells, have a high degree of plasticity, can be differentiated into M1-type (classical activation) and M2-type (selective-activated) macrophages in different environments, and the role of these two macrophages is more of a cloud of mud, Macrophages will differentiate into M2 type under the action of tumor microenvironment. Objective: To clarify whether magnesium isoglycyrrhizin significantly prolonged the survival of tumor-bearing mice: whether it was achieved by inducing the differentiation of macrophages to M1 in a tumor microenvironment. Methods: The micro-environment of gastric cancer was simulated with conditioned medium. The MFC cells of gastric cancer were treated with magnesium isoglycyrrhizin solution at different concentrations. The supernatant was then collected and added to the cultured mouse macrophages, RAW264.7, and the polarization of RAW264.7 was observed. The cells and supernatant of RAW264.7 were collected. The expression of iNOS, Arg1 and IL12 was detected by Western blot, and the expression of IL6, IL10, IL12, TGF-EMA1, TNF, VEGF-A and other factors related to tumor immunity were detected by ELISA, and the polarization of macrophages was analyzed. Results: The mouse macrophage RAW264.7 was divided into M2 type (high expression Arg1, low expression iNOS, IL12) by MFC cells of gastric cancer, and the supernatant of MFC cells of gastric cancer with magnesium isoglycyrrhizin was added to the culture medium, and the mouse macrophage RAW264.7 was induced to differentiate into M1 type (high expression of iNOS, IL12, The concentration of IL6, IL10, TGF-1, TNF-1, VEGF-A in the supernatant and RAW264.7 supernatant in DMEM complete culture medium were significantly increased after the supernatant of the untreated MFC cells was cultured for RAW264.7, while IL12 was significantly lower. The condition culture medium obtained after different concentration of magnesium isoglycyrrhizinate was added to MFC culture, and RAW264.7 was cultured. The concentration of IL6, IL10, TGF-1, TNF-1 and VEGF-A decreased significantly, and the concentration of IL12 increased. Conclusion: Magnesium isoglycyrrhizin can be induced indirectly by regulating the secretion of tumor cells, and can directly regulate the repolarization of tumor-related macrophages to M1-type macrophages in the microenvironment of gastric cancer, which is likely to be the role of magnesium isoglycyrrhizinate in the development of gastric cancer.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2

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