IL-27在艰难梭菌肠道感染中的免疫作用及机制研究

发布时间：2018-01-12 07:20

本文关键词：IL-27在艰难梭菌肠道感染中的免疫作用及机制研究　出处：《重庆医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:IL-27是调节宿主免疫应答的重要细胞因子,但在艰难梭菌感染中的免疫学作用尚不明确,因此本研究探讨了IL-27在艰难梭菌感染过程中的表达和发挥的作用及其作用机制。方法:采用ELISA检测艰难梭菌感染病人样本中IL-27的表达并分析IL-27与实验室检测指标的相关性;q-PCR和ELISA检测艰难梭菌刺激的DCs胞内IL-27 mRNA和上清中蛋白水平的表达;采用信号抑制剂处理DCs 24h,用ELISA检测上清中IL-27的表达变化,Western blot检测DCs胞内信号蛋白的磷酸化情况。用C57BL/6背景的IL-27R-/-鼠(KO)和野生鼠(WT)构建艰难梭菌感染模型,多方面检测感染效果后,检测CDI小鼠体内细胞因子的表达情况,并用重组IL-27治疗小鼠观察效应,通过检测细胞因子的表达、细胞吞噬功能的相关试验,对IL-27作用机制进行初步探究。此外,我们还检测了CDI患者和小鼠血清及肠组织中CXCL13的表达情况,并根据患者临床实验室检测指标进行回顾性分析,探讨CXCL13在CDI中的表达及临床意义。结果:CDI病人的血清和粪便中IL-27表达水平均升高,血清中IL-27水平与WBC呈正相关;艰难梭菌可激活DCs产生IL-27,在该菌刺激DCs后5min、30min时,P38MAPK、ERK、JNK、AKT、JAK、NF-κB的磷酸化显著增高。IL-27在艰难梭菌感染小鼠中表达水平显著升高;CDI后,与WT鼠相比,KO小鼠生存率低,体重下降早、幅度大,肠内容物艰难梭菌菌载量高,肠组织损伤严重,提示IL-27在CDI是可能起保护作用;直接用recombinant IL-27治疗CDI小鼠后发现,小鼠生存率提高,艰难梭菌感染症状得到了改善,这可能与治疗后IFN-γ升高有关,提示IFN-γ在CDI过程中有着至关重要的作用;IL-27预处理细胞24h后,检测细胞对艰难梭菌的吞噬作用,结果发现IL-27可增强PMs的吞噬活性,对中性粒细胞的吞噬活性无影响。此外,在CDI过程中高表达CXCL13,故可作为CDI诊断、患者预后评估或治疗CDI的潜在选择。结论:IL-27在艰难梭菌感中表达升高,并起保护作用,可治疗小鼠的CDI;另外,CDI过程中CXCL13高表达,可作为CDI诊断及患者预后的新标志物。
[Abstract]:Objective: IL-27 is an important cytokine to regulate the host immune response, but its immunological role in Clostridium difficulty infection is not clear. In this study, we investigated the expression, role and mechanism of IL-27 in the process of Clostridium difficulty infection. The expression of IL-27 was detected by ELISA and the correlation between IL-27 and laboratory indexes was analyzed. The expression of IL-27 mRNA and protein in supernatant of DCs stimulated by Clostridium difficulty was detected by q-PCR and ELISA. DCs was treated with signal inhibitor for 24 h and the expression of IL-27 in supernatant was detected by ELISA. Detection of intracellular signal protein phosphorylation in DCs by Western blot. IL-27R-P / R Ko and wild rat WT2 were used in C57BL / 6 background. To construct the model of Clostridium difficulty infection. The expression of cytokines in CDI mice was detected after various aspects of infection. The effect of recombinant IL-27 was observed in mice. The expression of cytokines was detected by detecting the expression of cytokines. In addition, we also detected the expression of CXCL13 in serum and intestinal tissues of CDI patients and mice. And according to the clinical laboratory test indicators of patients were analyzed retrospectively. To investigate the expression of CXCL13 in CDI and its clinical significance. Results the expression of IL-27 in serum and feces of patients with CDI increased, and the level of IL-27 in serum was positively correlated with WBC. Clostridium difficulty activated the production of IL-27 by DCs, and P38 MAPKK ERK ERK was activated to produce IL-27 at 30 min after stimulation of DCs. The phosphorylation of NF- 魏 B was significantly increased. IL-27 expression level was significantly increased in clostridium challenge infected mice. After CDI, the survival rate of KO mice was lower than that of WT mice, the weight loss was earlier, the amplitude was large, the intestinal contents of C. diffculent bacteria was high, and the intestinal tissue damage was serious. It is suggested that IL-27 may play a protective role in CDI. It was found that the survival rate of CDI mice was increased and the symptoms of Clostridium difficulty infection were improved after treatment with recombinant IL-27, which may be related to the increase of IFN- 纬 after treatment. It is suggested that IFN- 纬 plays an important role in the process of CDI. After pretreatment with IL-27 for 24 hours, the phagocytic activity of the cells against Clostridium difficulty was detected. The results showed that IL-27 could enhance the phagocytic activity of PMs, but had no effect on the phagocytic activity of neutrophils. The high expression of CXCL13 during CDI can be used as a potential choice for the diagnosis, prognosis evaluation or treatment of CDI in CDI. Conclusion the expression of 7% IL-27 is increased in the sense of Clostridium difficulty. It also plays a protective role in the treatment of CDI in mice. In addition, the high expression of CXCL13 in the process of CDI may be a new marker for the diagnosis and prognosis of CDI.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R446.5

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