白细胞介素-27抑制炎症作用的机制研究

发布时间：2018-05-08 18:42

本文选题：白细胞介素-27 + MDSCs　；参考：《浙江大学》2017年博士论文

【摘要】：本论文分为两个部分,分别研究了白细胞介素-27对肠炎相关肠癌的保护作用和白细胞介素-27对小鼠肥胖模型的调控作用及其机制。第一部分白细胞介素-27通过抑制肠上皮细胞分泌的促炎因子对肠炎相关肠癌的保护作用炎症和癌症的相关性一直是肿瘤免疫的研究重点之一,以往的结果表明,长期的炎性环境会加速肿瘤的形成。肠炎相关肠癌是一种典型的炎症相关肿瘤,长期的肠道炎症导致肠上皮的不断破坏和修复,长此以往加速了相关癌症相关基因的突变频率和肿瘤细胞的增殖。白细胞介素-27(Interleukin-27,IL-27)由p28和EBI3两个亚基组成,主要由活化的抗原递呈细胞分泌,其受体由gp130和WSX-1两个亚基组成,表达于多种免疫细胞、上皮细胞及内皮细胞表面。已有文献报道,IL-27在各种炎症相关的疾病中发挥调控功能,并且在不同疾病模型中针对不同靶细胞兼有抑炎和促炎两方面的作用。在肠炎的研究中,IL-27的功能也尚有争议,兼有其促进肠炎和抑制肠炎的报道,并且目前尚未有关于IL-27在肠炎相关肠癌中功能的报道。因此,本文通过建立由AOM/DSS诱导的肠炎相关肠癌(colitis-associated cancer)小鼠模型,研究IL-27对肠癌发生发展的作用,以期为炎症与癌症的关系研究提供相关的理论依据,为肿瘤微环境中的细胞因子的作用增添新的认识。我们首先用野生C57BL/6小鼠成功构建了 AOM/DSS模型,取小鼠结直肠肿瘤和癌旁组织,及健康小鼠结直肠组织,通过荧光定量PCR(Q-PCR)、Western Blot以及免疫荧光等手段检测到IL-27的两个亚基及其特异性受体亚基WSX-1在肿瘤组织局部表达升高,并且IL-27主要在肠道固有层细胞中表达。随后,我们使用IL-27R受体亚基WSX-1敲除的(WSX-1 KO)小鼠和野生(WT)小鼠同时构建AOM/DSS模型,结果发现与野生小鼠相比,WSX-1KO小鼠肠道肿瘤载量和数目明显增多,局部的肿瘤细胞增殖加强,并且伴随着促炎因子升高、肠道长度缩短等更严重的肠道炎症指标。为研究上述结果的机制,基于我们在WT和WSX-1 KO小鼠组织中观察到了不同的炎症程度,我们进一步使用流式细胞术检测了肠癌模型小鼠的肠道固有层细胞中的免疫细胞亚群的分布和变化,结果显示促肿瘤生长的髓系来源抑制性细胞(myeloid-derived suppressor cells,MDSCs)在 WSX-1 KO 小鼠的肠道中明显聚集增加,但其表面关键趋化因子受体CXCR2的表达并没有明显变化。我们进一步检测了肠上皮细胞中的趋化因子和促炎因子的表达,发现在WSX-1 KO肠癌模型小鼠的肠上皮中,除CXCR2的配体CXCL1的表达明显升高以外,促炎因子IL-6,TNF-α,GM-CSF的水平也有了明显升高,提示了 IL-27对肠上皮细胞可能发挥的抑炎作用。同时,免疫荧光实验也证明肿瘤局部CXCL1的下降与MDSCs数量的减少是一致的,提示肠上皮细胞来源的CXCL1增加可能是MDSCs数量增加的原因。随后我们在体外证明,使用中和抗体封闭了上皮细胞来源的CXCL1后,MDSCs的迁移减少;同时将IL-6和GM-CSF的中和抗体加入小鼠肠上皮来源的肿瘤细胞系CT26和MDSCs的共培养体系后,MDSCs的数量减少,证明肠上皮细胞来源的IL-6和GM-CSF促进MDSCs的存活。随后我们用荧光定量PCR和免疫荧光检测了 WT和WSX-1 KO肿瘤建模小鼠的肠道紧密连接蛋白的表达,发现没有明显统计学差异,并且血清中LPS也没有明显变化,证明促炎因子的变化不是由于局部来源于肠道菌群的病原相关模式分子(Pathogen-associated Molecular Pattern,PAMPs)的数量变化导致的,而可能直接由肠上皮细胞的反应性变化引起。因此我们进一步用LPS和IL-27在体外刺激了原代肠上皮细胞,发现IL-27可抑制LPS诱导的肠上皮细胞中促炎因子和趋化因子的分泌。最后,我们用抗生素组合喂食小鼠,清除肠道内微生物来源的PAMPs后,再用AOM/DSS诱导肿瘤模型,我们发现在清除了微生物来源的PAMPs后,WT和WSX-1KO模型小鼠肠道肿瘤、肠道固有层MDSCs数量、肠上皮细胞促炎因子和趋化因子的表达均无统计学差异,证明IL-27对肠炎相关肠癌的保护作用是依赖PAMPs的。综上,本部分研究证明在慢性肠炎相关的肠癌模型发病过程中,IL-27可以抑制PAMPs触发的肠上皮细胞中促炎因子和趋化因子的表达,从而降低了促肿瘤的髓系来源抑制细胞(MDSCs)的聚集,因此对肿瘤的发生发展起到抑制作用。本研究为阐明肿瘤微环境中细胞因子的调控作用增添了新的理论依据,也可能为肠癌的免疫治疗提供新的思路。第二部分白细胞介素-27受体敲除促进小鼠肥胖及其机制的初步研究肥胖是现代社会危害人类健康的隐形杀手之一,它所引发的代谢病症长久以来困扰着人们的身体健康。我们对脂肪功能的认识,也逐渐从简单存储能量,到作为内分泌系统的一部分调节人体代谢功能。内脏脂肪的含量和状态,对健康有着非常重要的影响。随着研究的深入,肥胖被认为是一种慢性炎症状态,脂肪组织,特别是内脏脂肪组织(visceral adipose tissue,VAT)分泌的各种促炎因子如IL-6、TNF-a和C-反应蛋白(C-reactiveprotein,CRP)等在肥胖状态下均表达上升,导致胰岛素抵抗等相关表现。近年来发现,内脏脂肪组织中存在一种特殊的调节性T细胞(Treg),脂肪Treg通过分泌IL-10和TGF-β对脂肪代谢的稳态起到重要调节作用。正常状态Treg细胞在脂肪中聚集,VAT Treg占CD4+ T细胞的比例高于其他淋巴器官和非淋巴器官,而在肥胖状态下,Treg细胞比例在脂肪组织中急剧下降。在进行第一部分实验的时候,我们发现在较大年龄(24周以上)的小鼠中,WSX-1 KO小鼠体重略高于WT小鼠,同时查阅文献发现以往并没有IL-27在肥胖中的作用研究,因此进行了第二部分实验。WSX-1 KO和WT小鼠给予正常饮食饲养在同样环境下24周后,我们首先检测其体重变化,发现WSX-1KO小鼠体重相较于WT小鼠有明显增加,并且内脏脂肪的重量也有明显上升,流式检测结果表明WSX-1 KO小鼠中VATTreg数量下降。这些结果提示了 IL-27受体敲除可促进小鼠肥胖。随后,我们进一步用高脂饲料诱导肥胖模型,得到同样结果。在喂养过程中,虽然两组进食量并没有差距,但是WSX-1 KO小鼠的体重高于WT小鼠,核磁共振结果显示其体脂含量也高于WT小鼠,并且WSX-1 KO小鼠中VAT Treg数量也显著降低,VAT Treg的相关转录因子Foxp3和PPAR-γ mRNA水平在内脏脂肪中的含量也显著下降,这些结果进一步证明,在高脂饮食诱导的肥胖模型中,IL-27/WSX-1信号也可以对肥胖发挥抑制作用。为了解这种现象是否具有临床相关性,我们进一步用ELISA检测了肥胖患儿血浆中的IL-27含量,但有趣的是,发现跟对照组相比,肥胖儿童血浆中IL-27含量升高,我们推测IL-27可能是以一种代偿效应发挥作用,在肥胖状态下应激分泌。具体机制还有待于进一步深入研究。综上所述,本部分研究我们初步发现了 IL-27/WSX-1信号缺失后可促进小鼠肥胖的表型,首次在小鼠肥胖模型中阐述了 IL-27可能发挥的抑炎作用,希望在日后的研究中,可以为肥胖及相关并发症的机制研究及其治疗提供理论依据和潜在靶点。
[Abstract]:This thesis is divided into two parts. The protective effect of interleukin -27 on colonic cancer and the regulation of interleukin -27 on the model of obesity in mice and its mechanism. The first part is the protective action of interleukin -27 on the protection of intestinal inflammation and cancer by inhibiting the proinflammatory factors secreted by intestinal epithelial cells Correlation has been one of the key points in the study of tumor immunity. Previous results showed that a long-term inflammatory environment accelerated the formation of tumors. Enteritis related colon cancer is a typical inflammation related tumor. Long term intestinal inflammation leads to the continuous destruction and repair of intestinal epithelium, which has accelerated the mutation frequency of related cancer related genes. The proliferation of tumor cells. Interleukin -27 (Interleukin-27, IL-27) is composed of two subunits of p28 and EBI3, mainly secreted by activated antigen presenting cells. Their receptors are composed of two subunits of gp130 and WSX-1, expressed in various immune cells, epithelial cells and endothelial cells. It has been reported that IL-27 is in various inflammatory related diseases. The role of IL-27 is also controversial in the study of enteritis, and there are also reports on the promotion of enteritis and inhibition of enteritis in the study of enteritis, and there is no report on the function of IL-27 in colitis related colon cancer. To establish a AOM/DSS induced colitis-associated cancer mouse model to study the effect of IL-27 on the development of colon cancer in order to provide a theoretical basis for the study of the relationship between inflammation and cancer, and to add a new understanding to the role of cytokine in the tumor microenvironment. We first succeeded in using wild C57BL/6 mice. The AOM/DSS model was constructed to take the colorectal tumor and para cancerous tissue in mice and the colorectal tissue in healthy mice. The expression of two subunits and specific receptor subunits WSX-1 in the tumor tissues was increased by fluorescence quantitative PCR (Q-PCR), Western Blot and immunofluorescence, and IL-27 was mainly in the intestinal lamina propria cells. Then, we used the IL-27R receptor subunit WSX-1 knockout (WSX-1 KO) mice and the wild (WT) mice to construct the AOM/DSS model simultaneously. The results showed that compared with the wild mice, the intestinal tumor loading and number of WSX-1KO mice increased significantly, the local tumor cell proliferation was stronger, and the proinflammatory factors were increased and the length of the intestine was shortened. A more serious indicator of intestinal inflammation. For the study of the above results, based on the different levels of inflammation observed in WT and WSX-1 KO mice, we further used flow cytometry to detect the distribution and changes in the immune cell subsets in the intestinal lamina propria cells of the colon cancer model mice. The results showed that the tumor growth was promoted. Myeloid-derived suppressor cells (MDSCs) was significantly increased in the intestinal tract of WSX-1 KO mice, but the expression of the key chemokine receptor CXCR2 on the surface was not significantly changed. We further detected the expression of chemokines and proinflammatory factors in the intestinal epithelial cells, and found that the WSX-1 KO colon cancer model was found. In the intestinal epithelium of the mice, the levels of the proinflammatory factor IL-6, TNF- a, and GM-CSF were also significantly elevated in addition to the increase in the expression of the CXCR2 ligand CXCL1, suggesting the possible anti-inflammatory effect of IL-27 on intestinal epithelial cells. At the same time, the immunofluorescence test also showed that the decrease of local CXCL1 in the tumor was consistent with the decrease in the number of MDSCs. The increase of CXCL1 in the intestinal epithelial cells may be the cause of the increase in the number of MDSCs. Subsequently, we demonstrated in vitro that the use of neutralizing antibodies closed the CXCL1 of epithelial cells and reduced the migration of MDSCs; meanwhile, IL-6 and GM-CSF neutralizing antibodies were added to the co culture system of the murine epithelial cells of the intestinal epithelium, CT26 and MDSCs, MDSCs. The number of IL-6 and GM-CSF derived from intestinal epithelial cells promoted the survival of MDSCs. Then we detected the expression of close connexin in the intestinal tract of WT and WSX-1 KO tumor modeling mice by fluorescence quantitative PCR and immunofluorescence, and found no significant difference in statistics, and there was no significant change in serum LPS. The change is not due to the changes in the number of Pathogen-associated Molecular Pattern (PAMPs) derived from the intestinal flora, which may be caused by the reactive changes in the intestinal epithelial cells. Therefore, we further stimulated the primary intestinal epithelial cells with LPS and IL-27 in vitro, and found that IL-27 inhibits LPS. The secretion of pro-inflammatory factors and chemokines in the intestinal epithelial cells was induced. Finally, we fed the mice with antibiotic combination, removed the PAMPs in the intestinal tract and induced the tumor model with AOM/DSS. We found that after the removal of PAMPs from the microbial sources, we found the intestinal tumor in the WT and WSX-1KO model mice, the amount of the intestinal lamina propria MDSCs. There is no statistical difference in the expression of proinflammatory and chemokines in intestinal epithelial cells. It is proved that the protective effect of IL-27 on enterocolitis is dependent on PAMPs. In this part, this part of this study proved that IL-27 could inhibit the proinflammatory factors and chemokines in the intestinal epithelial cells triggered by PAMPs during the pathogenesis of chronic enteritis. Expression, thus reducing the accumulation of myeloid source inhibition cells (MDSCs), can inhibit the development of tumor. This study provides a new theoretical basis for clarifying the regulation of cytokines in tumor microenvironment, and may also provide new ideas for the immunotherapy of colon cancer. The second part of interleukin -27 Receptor knockout promotes obesity and its mechanism in mice. Obesity is one of the invisible killer of human health in modern society. Its metabolic disorder has been perplexing people's health for a long time. Our understanding of fat function is gradually from storing energy simply to regulating human body as part of the endocrine system. Metabolic function. The content and state of visceral fat have a very important impact on health. As the study goes deep, obesity is considered a chronic inflammatory state, and the various proinflammatory agents secreted by visceral adipose tissue (VAT), especially the visceral adipose tissue, such as IL-6, TNF-a, and C- reactive protein (C-reactiveprotein, CRP), are secreted. In recent years, a special regulatory T cell (Treg) has been found in visceral adipose tissue. Fat Treg plays an important role in the homeostasis of fat metabolism by secreting IL-10 and TGF- beta. Treg cells in normal state are aggregated in fat and VAT Treg accounts for CD4+ T. In the first part of the experiment, we found that WSX-1 KO mice were slightly higher than the WT mice at a large age (more than 24 weeks), and the literature found no IL-27 in the past. The role of obesity was studied, so second parts of the experiment.WSX-1 KO and WT mice were given normal diet in the same environment for 24 weeks. We first detected the body weight changes. It was found that the weight of WSX-1KO mice increased significantly compared to the WT mice, and the weight of visceral fat increased significantly, and the flow test results showed WSX-1 KO. The number of VATTreg in mice decreased. These results suggest that IL-27 receptor knockout can promote obesity in mice. Then, we further use high fat diet to induce obesity model and get the same results. In the feeding process, although there is no difference in the intake of two groups, the weight of the WSX-1 KO mice is higher than that of the WT mice, and the MRI results show the body fat. The content was also higher than that of WT mice, and the number of VAT Treg in the WSX-1 KO mice decreased significantly, and the level of VAT Treg related transcription factors Foxp3 and PPAR- gamma mRNA decreased significantly in visceral fat. These results further demonstrated that the IL-27/WSX-1 signal could also inhibit obesity in the high fat diet induced obesity model. In order to understand the clinical relevance of this phenomenon, we further detected the IL-27 content in the plasma of obese children with ELISA, but it is interesting to find that the plasma levels of IL-27 in obese children are higher than those in the control group. We speculate that IL-27 may play a role in a compensatory effect and stress secretion in the obese state. For the first time, we have discovered the phenotype that can promote the obesity in mice after the deletion of IL-27/WSX-1 signal. We first described the possible anti inflammatory effect of IL-27 in the model of obesity in mice. We hope that in the future study, we can study the mechanism of obesity and related complications and the mechanism in the future study. The treatment provides theoretical basis and potential targets.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R730.3

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