西咪替丁调控免疫反应的研究

发布时间：2018-10-31 11:29

【摘要】：免疫的调控对于宿主的免疫应答至关重要。调节性T细胞(regulatory T cell,简称Treg cell)主要参与外周免疫耐受并负向调控免疫反应以防止过度免疫反应及自身免疫等对宿主本身造成的损害。西咪替丁(Cimetidine,简称CIM)是一种组胺的H2受体拮抗剂,最初发现能够降低胃酸分泌以及消化道溃疡,近年来发现西咪替丁对于免疫反应也有着多种调控效应。本实验室已有的研究发现,CIM可作为疫苗佐剂对适应性免疫反应起到促进作用,进而增强疫苗的免疫效果,进一步结果显示其对调节性T细胞功能可能有抑制作用。然而,CIM的这种调节作用的分子机理并不十分清楚。本研究第一部分针对CIM在体外如何在分子水平上对调节性T细胞的影响进行了研究；而第二部分是研究CIM在体内先天性免疫系统中对炎性反应的调控作用。第一部分体外研究：用CIM体外刺激人的CD4+CD25+hTreg细胞和JurkatT细胞,并采用蛋白免疫印迹技术分析了调节性T细胞的关键功能转录因子Foxp3的表达和稳定性的变化,证明其影响主要是发生在蛋白质稳定水平。进一步通过沉默E3泛素化连接酶Stub1,分析了CIM对Stubl活化的影响。结果显示,CIM能够激活Stubl并引起Foxp3的泛素化降解,从而证明CIM影响Foxp3的功能主要是通过降解Foxp3的蛋白质活性而达到的。上游PI3K-Akt-mTOR信号通路是CIM作用的主要通路,利用抑制剂LY294002封闭PI3K-Akt-mTOR信号通路证明,CIM对Foxp3的降解是依赖于PI3K-Akt-mTOR信号通路激活。由此表明,CIM可通过激活PI3K-Akt-mTOR信号通路而促进E3泛素化连接酶Stubl所介导的Foxp3降解,从而降低了Treg细胞的免疫抑制功能,一定程度上可促进适应性免疫应答。第二部分体内研究：炎症性肠炎(inflammatory bowel disease,简称IBD)是一类由肠道微生物致病菌以及炎症反应共同作用所引起的肠道疾病,其发病原因复杂、易反复,长期患病易转化为结肠直肠癌。已有研究表明,CIM对胃溃疡及消化道溃疡有治疗作用,能够通过降低先天性免疫反应中一些炎症因子的产生。本研究在使用TNBS诱导溃疡型炎症性肠道炎的过程中发现,注射一次CIM能够有效地减轻小鼠患IBD的病症,进而保护小鼠免于IBD所致的死亡。免疫组化检测结果发现,在溃疡性IBD模型初期的炎症反应中,巨噬细胞起着重要的作用,其浸润的数量以及表达的TNF-α等相关炎症分子,能够被CIM有效的抑制。进一步采用小鼠原代骨髓来源巨噬细胞(BMDM)的试验证明,CIM能够显著降低由LPS激活的BMDM中相关炎症因子TNF-a, IL-6, IL-1β等的转录,并降低TNF-α的水平。蛋白免疫印迹分析结果表明,CIM能够通过抑制受到刺激信号激活的NF-κB通路,进而抑制相关炎症因子基因的表达。经细胞过继转移试验和荧光定量PCR分析,表明CIM可能对于巨噬细胞向炎症部位的迁移有着一定的抑制作用。综上我们的研究结果表明,CIM可降低适应性免疫反应中Treg细胞的免疫抑制功能；在溃疡性炎症反应中,CIM在先天性免疫反应初期可通过抑制NF-κB信号通路的激活而降低相关炎症因子的表达,从而保护小鼠免于因过激的炎症反应而直接导致的死亡。
[Abstract]:The regulation of immunity is critical to the immune response of the host. Regulatory T cell, called Treg cell, is mainly involved in peripheral immune tolerance and negative regulation of immune response to prevent over-immune response and autoimmune damage to the host itself. Cimetidine (CIM) is a histamine H2 receptor antagonist, initially found to be able to reduce gastric acid secretion and digestive tract infection. In recent years, it has been found that there are a variety of regulatory effects on the immune response. It has been found in this laboratory that CIM can promote the adaptive immune response as a vaccine adjuvant, thus enhancing the immune effect of the vaccine, and further shows that it may have an inhibitory effect on the regulatory T cell function. However, the molecular mechanism of this regulation of CIM is not very clear. In the first part of this study, the effects of CIM on regulatory T cells in vitro were studied, and the second part was to investigate the effects of CIM on inflammatory response in vitro. In vitro, the expression and stability of the transcription factor Foxp3 of CD4 + CD25 + hTreg cells and Jurkat T cells stimulated by CIM in vitro were studied. The effect of CIM on Stubl activation was further analyzed by silencing E3 ubiquitin ligase Stub1. The results show that CIM can activate Stubl and induce ubiquitination degradation of Foxp3, thus demonstrating that the function of CIM on Foxp3 is mainly achieved by degrading the protein activity of Foxp3. The upstream PI3K-Akt-mTOR signaling pathway is the main pathway for CIM, and the degradation of the CIM to Foxp3 is dependent on the activation of the PI3K-Akt-mTOR signaling pathway by using the inhibitor LY294002 to close the PI3K-Akt-mTOR signaling pathway. Thus, CIM can promote the degradation of Foxp3 mediated by E3 ubiquitin ligase Stubl by activating the T-Akt-mTOR signaling pathway, thus reducing the immunosuppressive function of Treg cells, which can promote adaptive immune response to a certain extent. In the second part, inflammatory bowel disease (IBD) is a kind of intestinal disease caused by intestinal microorganism pathogenic bacteria and inflammatory response. The cause of the disease is complicated, it is easy to repeat, and the long-term illness is easy to be converted into colorectal cancer. It has been shown that CIM has therapeutic effect on the digestive tract and digestive tract, and can reduce the generation of some inflammatory factors in the innate immune response. In the process of using TNBS to induce inflammatory bowel inflammation, it was found that the injection of CIM could effectively reduce the symptoms of IBD in mice and protect the mice from the death caused by IBD. The results of immunohistochemistry show that macrophages play an important role in inflammatory response in the early stage of IBD model, the number of infiltration and the expression of TNF-CoV and other relevant inflammatory molecules can be effectively inhibited by CIM. Further use of mouse primary bone marrow-derived macrophages (BMDM) demonstrated that CIM significantly reduced the transcription of TNF-a, IL-6, IL-1, etc. associated with LPS-activated BMDM, and reduced TNF-alpha levels. The results of Western blot analysis showed that CIM could inhibit the expression of the related inflammatory factor gene by inhibiting the NF-Sepharose B pathway stimulated by the stimulation signal. Through cell adoptive transfer assay and fluorescence quantitative PCR analysis, it is shown that CIM may have a certain inhibitory effect on the migration of macrophages to inflammatory sites. The results of our study indicate that CIM can reduce the immunosuppressive function of Treg cells in adaptive immune response; in inflammatory response, CIM can reduce the expression of relevant inflammatory factors by inhibiting the activation of NF-Sepharose B signaling pathway at the beginning of the innate immune response. thereby protecting the mice from death caused by excessive inflammatory reactions.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R392

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