異甘草酸鎂調(diào)節(jié)胃癌炎性微環(huán)境的抑癌機(jī)制研究
發(fā)布時(shí)間：2019-06-09 16:14
【摘要】：腫瘤的發(fā)生、發(fā)展和侵襲轉(zhuǎn)移與炎癥密切相關(guān),免疫細(xì)胞和炎癥因子幾乎影響到腫瘤進(jìn)展的每一方面。腫瘤相關(guān)性炎癥已被列入腫瘤的第七種特征,腫瘤相關(guān)炎癥能夠通過促進(jìn)血管新生、促進(jìn)腫瘤轉(zhuǎn)移、抑制抗腫瘤免疫應(yīng)答及改變腫瘤細(xì)胞對(duì)化療藥的敏感性等方面促進(jìn)腫瘤的發(fā)生和進(jìn)展,而這種作用主要依賴腫瘤微環(huán)境(TME)實(shí)現(xiàn)。因此調(diào)節(jié)其炎性微環(huán)境可能會(huì)翻開腫瘤治療策略的新一頁(yè)。胃癌作為世界第五大常見的惡性腫瘤,發(fā)現(xiàn)晚,治療難,考慮其與幽門螺桿菌(Hp)感染和慢性胃炎關(guān)聯(lián)甚密,我們?cè)噲D找到通過調(diào)節(jié)其炎性微環(huán)境控制腫瘤的方法。臨床常用藥異甘草酸鎂(MgIG)被發(fā)現(xiàn)的抗癌療效為我們提供了思路。異甘草酸鎂是甘草酸的第四代制劑,具有抗炎保肝、抗病毒、抗氧化、抗過敏、調(diào)節(jié)免疫和抗癌療效。課題組也在前期工作中通過動(dòng)物實(shí)驗(yàn),驗(yàn)證了異甘草酸鎂對(duì)胃癌的干預(yù)作用,在此基礎(chǔ)上,我們對(duì)小鼠腫瘤組織中炎性細(xì)胞因子表達(dá)情況和炎性細(xì)胞密度進(jìn)行了檢測(cè),在對(duì)此結(jié)果進(jìn)行分析討論的基礎(chǔ)上,進(jìn)一步選取巨噬細(xì)胞作為研究對(duì)象,體外模擬腫瘤的炎性微環(huán)境,觀察異甘草酸鎂對(duì)微環(huán)境中巨噬細(xì)胞極化的影響,以此探討異甘草酸鎂是否通過調(diào)節(jié)胃癌炎性微環(huán)境從而干預(yù)胃癌。第一部分異甘草酸鎂對(duì)胃癌小鼠微環(huán)境中炎癥細(xì)胞因子表達(dá)和炎性細(xì)胞的影響研究背景:已知異甘草酸鎂處理皮下荷瘤的小鼠后小鼠瘤重減輕,瘤體積縮小,生存時(shí)間延長(zhǎng)。同時(shí)已知異甘草酸鎂對(duì)炎癥因子和炎性細(xì)胞的調(diào)節(jié)作用,以及胃癌的炎性微環(huán)境對(duì)胃癌進(jìn)展的不可忽視的作用。目的:檢測(cè)胃癌小鼠腫瘤組織中免疫細(xì)胞和炎癥因子,發(fā)現(xiàn)異甘草酸鎂是否通過對(duì)胃癌炎性微環(huán)境的調(diào)節(jié)來抑制胃癌的發(fā)展。方法:首先我們選擇了一款能檢測(cè)96個(gè)與炎癥和自身免疫相關(guān)的分子表達(dá)水平的轉(zhuǎn)錄組芯片來檢測(cè)異甘草酸鎂對(duì)小鼠胃癌移植瘤組織內(nèi)這些重要的細(xì)胞因子的表達(dá)水平的影響,主要為趨化因子家族及受體、白介素家族及受體以及NF κB通路關(guān)鍵分子,并用qPCR對(duì)芯片結(jié)果進(jìn)行了驗(yàn)證。然后我們用免疫組化對(duì)腫瘤微環(huán)境中的關(guān)鍵炎性細(xì)胞進(jìn)行染色。結(jié)果:異甘草酸鎂下調(diào)了小鼠胃癌組織中趨化因子及其受體CCL3、CCL5、CCL8、CCL11、CCL13、CCL19、CCL21、CXCL1、CXCL2、CXCL8、CCR1、CCR3、CCR4、CCR7、CXCR1、CXCR2的表達(dá);下調(diào)了小鼠胃癌組織中TLRs、MyD88、Tollip、NF-κb的表達(dá);下調(diào)了小鼠胃癌組織中il1a、il1b、il1r1、il1rap、il1rn、il5、il6、il6r、il10、il10rb、il15、il17a、il18、il22、il23a、il23r的表達(dá),上調(diào)了il2、il12的表達(dá);降低了小鼠胃癌組織中treg細(xì)胞密度和活性,增大了胃癌組織中cd8+t細(xì)胞密度,增大了胃癌組織中巨噬細(xì)胞的密度(p0.05)。結(jié)論:異甘草酸鎂可能通過調(diào)節(jié)小鼠胃癌組織中趨化因子及其受體、白介素家族及受體以及nf κb通路關(guān)鍵分子的表達(dá)和treg細(xì)胞、cd8+t細(xì)胞的密度(和活性)而抑制胃癌的發(fā)展。第二部分異甘草酸鎂對(duì)胃癌微環(huán)境中巨噬細(xì)胞極化的誘導(dǎo)作用研究背景:第一部分我們發(fā)現(xiàn)CD68+的巨噬細(xì)胞密度在異甘草酸鎂用藥組增大,而現(xiàn)在普遍認(rèn)為巨噬細(xì)胞密度越高,預(yù)后越差(胰腺癌中除外),因此異甘草酸鎂用藥組巨噬細(xì)胞密度增加但小鼠生存期延長(zhǎng)似乎與以往文獻(xiàn)不符。但是巨噬細(xì)胞是一個(gè)異質(zhì)的細(xì)胞群,具有高度的可塑性,在不同的環(huán)境下可分化為M1型(經(jīng)典激活)和M2型(選擇性激活)巨噬細(xì)胞,而這兩種巨噬細(xì)胞的作用更是云泥之別,巨噬細(xì)胞在腫瘤微環(huán)境的作用下會(huì)分化為M2型。目的:闡明異甘草酸鎂明顯延長(zhǎng)荷瘤小鼠生存期的機(jī)制:是否是通過誘導(dǎo)腫瘤微環(huán)境中巨噬細(xì)胞向M1分化實(shí)現(xiàn)的。方法:采用條件培養(yǎng)基模擬胃癌的微環(huán)境,先用不同濃度的異甘草酸鎂溶液處理小鼠胃癌MFC細(xì)胞,然后收集上清,加到培養(yǎng)的小鼠巨噬細(xì)胞RAW264.7中,觀察RAW264.7的極化情況,再分別收集RAW264.7細(xì)胞和上清,細(xì)胞提取蛋白western blot法檢測(cè)iNOS、Arg1、IL12表達(dá)水平,上清ELISA法檢測(cè)IL6、IL10、IL12、TGF-β1、TNF、VEGF-A等與腫瘤免疫密切相關(guān)因子,從而分析巨噬細(xì)胞極化情況。結(jié)果:胃癌MFC細(xì)胞可以誘導(dǎo)小鼠巨噬細(xì)胞RAW264.7分化為M2型(高表達(dá)Arg1,低表達(dá)iNOS、IL12),而在培養(yǎng)基中加入異甘草酸鎂培養(yǎng)的胃癌MFC細(xì)胞上清,則誘導(dǎo)小鼠巨噬細(xì)胞RAW264.7分化為M1型(高表達(dá)iNOS、IL12,低表達(dá)Arg1);而未處理的MFC細(xì)胞上清培養(yǎng)RAW264.7后,上清中的IL6、IL10、TGF-β、TNF-α、VEGF-A,較DMEM完全培養(yǎng)基培養(yǎng)的RAW264.7上清中濃度顯著升高,而IL12明顯要低。在MFC培養(yǎng)上清中加入不同濃度異甘草酸鎂后獲得的條件培養(yǎng)基,再去培養(yǎng)RAW264.7,其IL6、IL10、TGF-β、TNF-α、VEGF-A濃度顯著降低,IL12濃度升高。結(jié)論:異甘草酸鎂可以通過調(diào)節(jié)腫瘤細(xì)胞的分泌間接誘導(dǎo),和直接調(diào)控胃癌微環(huán)境中腫瘤相關(guān)巨噬細(xì)胞向M1型巨噬細(xì)胞復(fù)極化,這極有可能是異甘草酸鎂干預(yù)胃癌發(fā)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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.2
本文編號(hào)：2495693
[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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.2
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