RegⅢγ在乳酸菌拮抗结肠炎黏膜免疫中的作用研究

发布时间：2018-04-07 15:05

本文选题：乳酸菌　切入点：RegIIIγ　出处：《吉林农业大学》2015年硕士论文

【摘要】：肠道上皮是机体内最大的黏膜屏障,其产生的抑菌因子胰岛再生源蛋白IIIγ(regenerating islet-derived protein IIIγ,RegIIIγ)是肠道先天黏膜免疫中重要成分。RegIIIγ为一种可以直接杀菌的C型凝集素,研究发现髓样分化因子88(myeloid differentiation factor88,MyD88)及IL-23/IL-22等因子的产生对RegIIIγ的表达其促进作用。健康肠道内RegIIIγ具有较强的杀菌能力,在防止共生菌和致病菌穿透肠道屏障及炎症性肠病(Inflammatory Bowel Disease,IBD)发病机制中发挥重要作用。乳酸菌作为肠道黏膜表面优势菌群具有诸多益生功能,可通过与肠道上皮相互作用调节肠道内环境稳定及加强免疫防御,从而促进肠道健康,同时在IBD的防治方面也具有一定疗效。然而乳酸菌在IBD中的作用机理仍不明确,尤其在乳酸菌与肠道上皮细胞相互作用过程中对抑菌蛋白RegIIIγ调控机制尚不清晰。本研究通过分析乳酸菌干预的小鼠机体内RegIIIγ的量的变化,初步探讨RegIIIγ在乳酸菌免疫防御及拮抗肠炎黏膜免疫中的作用及分子机制。研究内容如下:(1)混合乳酸菌对健康小鼠肠道RegIIIγ的量的影响本实验利用前期已筛选出的具有较强益生活性的混合乳酸菌干预健康小鼠,通过肠道HE染色、荧光定量PCR、免疫荧光及酶联免疫吸附试验技术检测干预前后小鼠肠道组织结构变化、Reg IIIγ及细胞因子包括MyD88、IL-23(IL-12p40,IL-23p19)、IL-22、IL-17A及TNF-α的量的变化,初步探索在正常肠道内乳酸菌对肠道黏膜先天免疫因子RegIIIγ及细胞因子调控的分子机制。此外,利用灭活菌及菌液培养上清干预正常小鼠,通过Q-PCR技术检测干预前后小鼠肠道内RegIIIγmRNA水平的变化,分析混合乳酸菌所分泌的代谢产物对肠道中RegIIIγ的影响。结果表明:混合乳酸菌定植正常小鼠肠道后RegIIIγ的量显著增高,与对正常照组相比具有显著性差异。组织学分析显示,乳酸菌定植后肠道组织结构正常,无炎症产生。且混合乳酸菌干预14d后,小鼠肠道中MyD88、IL-23及IL-22的量显著增高;而IL-17A及TNF-α略有升高,但与对照组无显著差异。此外混合乳酸菌干预的小鼠肠道中,仅活菌干预组和热灭活菌干预组中RegIIIγ的量显著提高,而乳酸菌培养上清组与对照组相比无显著性差异。(2)混合乳酸菌诱导的Reg IIIγ在细菌性结肠炎黏膜免疫中的作用研究本实验首先利用啮齿类柠檬酸杆菌(C.rodentium)感染健康小鼠建立肠炎模型,并应用不同剂量(1×109cfu及1×1010cfu)混合乳酸菌对肠炎小鼠进行干预。混合乳酸菌组干预14d,每天监测各组小鼠体重变化。乳酸菌干预7d及14d处死小鼠,取出各组结肠内容物及肠段进行C.rodentium定植量的测定及结肠病理组织学检测,同时应用Q-PCR、IF及ELISA检测RegIIIγ及细胞因子MyD88、IL-23(IL-12p40,IL-23p19)、IL-22、IL-17A及TNF-α表达量的变化,初步分析在C.rodentium所致肠炎中混合乳酸菌的作用。结果表明,肠炎模型组随时间推移机体体重显著降低,同时结肠中C.rodentium定植量显著增多,结肠组织结构严重破坏。而经混合乳酸菌干预后C.rodentium结肠内定植量显著下调,小鼠体重呈恢复趋势,结肠病变得到显著改善,其中以1×1010cfu干预组效果最为显著。与正常对照组相比,肠炎模型组小鼠结肠RegIIIγ及细胞因子MyD88、IL-23、IL-22、IL-17A及TNF-α显著上调并随着炎症程度的增高逐渐升高。而混合乳酸菌干预组与肠炎模型组相比RegIIIγ及细胞因子MyD88、IL-23、IL-22、IL-17A及TNF-α表达量显著降低,并随着干预天数的增加表达水平进一步下调,其中以1×1010cfu剂量干预组对RegIIIγ及相关因子的影响最为显著。综上所述,乳酸菌的自身成分或其自身结构而并非分泌的代谢产物可在一定程度上引起MyD88、IL-23及IL-22的升高继而促进RegIIIγ增多。且乳酸菌的定植有助于降低机体炎性反应,恢复机体免疫平衡以达到缓解肠炎的作用。进一步研究表明乳酸菌缓解IBD的作用机制可能与降低RegIIIγ异常的表达有关。
[Abstract]:Is the largest intestinal mucosal barrier in the body, then the islet inhibitory factor III protein gamma (regenerating islet-derived source protein III y, RegIII y.RegIII y) is an important component of innate mucosal immunity in the gut as a direct bactericidal C type lectin, the research found that myeloid differentiation factor 88 (myeloid differentiation factor88 MyD88, IL-23/IL-22) and cytokines on the expression of RegIII gamma. Its role in promoting the healthy intestine RegIII gamma has strong sterilization ability, in the prevention of commensal and pathogenic bacteria penetrate the intestinal barrier and inflammatory bowel disease (Inflammatory Bowel, Disease, IBD) play an important role in the pathogenesis of intestinal mucosal surface. The lactic acid bacteria as advantage bacteria have many probiotic functions, with intestinal epithelial interactions regulate intestinal homeostasis and enhance the immune defense, so as to promote intestinal health, with In the prevention and treatment of IBD have a certain effect. However, lactic acid bacteria in the IBD mechanism is not clear, especially in the process of lactic acid bacteria and intestinal epithelial cells in the interaction of RegIII inhibitory protein gamma control mechanism is not clear. The changes of mice through analysis of lactic acid bacteria in the intervention of RegIII gamma amount preliminary study of RegIII gamma, lactic acid bacteria in immune defense and antagonistic enteritis mucosal immunity function and mechanism. The contents are as follows: (1) effect of mixed lactic acid bacteria on healthy mice intestinal RegIII gamma quantity has strong activity of probiotic mixed lactic acid bacteria in this experiment using pre screened intervention mice through the intestinal tract HE staining, fluorescence quantitative PCR, immunofluorescence and ELISA structural changes of intestinal tissue in mice were detected before and after intervention test technology, Reg III gamma and cytokines including MyD88, IL-23 (IL-12p40, IL-23p 19), IL-22, IL-17A and TNF- changes in the amount of alpha, preliminary exploration on normal intestinal lactic acid bacteria on intestinal mucosal innate immune factor RegIII gamma and cytokine regulation mechanism. In addition, the supernatant intervention in normal mice using inactivated bacteria and bacteria, through the change of mouse intestinal RegIII gamma mRNA levels before and after the intervention of Q-PCR technology, analysis of the effect of metabolites secreted by the mixture of lactic acid bacteria on intestinal RegIII gamma. The results showed that the mixture of lactic acid bacteria in normal mouse intestine after RegIII gamma were significantly higher compared with the normal control group, with significant difference. Histological analysis showed that the lactic acid bacteria of intestinal structure is normal. No inflammation. And, mixed lactic acid bacteria 14d treated MyD88 mice in the gut, IL-23 and IL-22 were significantly increased; while IL-17A and TNF- alpha was slightly increased, but no significant difference with the control group. In addition to mixed lactic acid bacteria Intestinal intervention, only viable intervention group and heat inactivated bacteria in the intervention group RegIII gamma were significantly increased, and lactic acid bacteria culture supernatant group and the control group had no significant difference. (2) effect of Reg induced by III mixture of lactic acid bacteria in bacterial colitis mucosal immunity in the research the first use of rodent Citrobacter (C.rodentium) infection in healthy mice colitis model, and the application of different doses (1 * 109cfu and 1 * 1010cfu) mixed lactic acid bacteria to intervene enteritis mice. Mixed lactic acid bacteria 14d intervention, body weight changes of mice were monitored daily. The lactic acid bacteria 7d and 14d intervention mice were killed remove the determination and colonic histopathology were detected the contents of the colon and intestinal C.rodentium colonization rate, while application of Q-PCR, detection of MyD88 RegIII gamma and cytokines IF and ELISA, IL-23 (IL-12p40, IL-23p19), IL-22, expression of IL-17A and TNF- alpha The amount of change, a preliminary analysis of mixed lactic acid bacteria in C.rodentium induced enteritis in rats. Results showed that the rats in the model group with the passage of time the body weight decreased significantly, while C.rodentium in colon colonization rate increased significantly, the colon tissue destruction of structure. And the mixed lactic acid bacteria after intervention C.rodentium colon colonization was significantly reduced, the body weight in mice recovery trend, colonic lesions improved significantly, with 1 * 1010cfu group had the best effect. Compared with the normal control group, the rats in the model group colon RegIII mouse gamma and cytokines MyD88, IL-23, IL-22, IL-17A and TNF- significantly increased with the increase of alpha and the degree of inflammation was gradually increased. While the mixed lactic acid intervention group the rats in the model group compared with RegIII gamma and cytokines MyD88, IL-23, IL-22, IL-17A and the expression of TNF- alpha was significantly reduced with the increase in the number of levels of further intervention The cut, with 1 * 1010cfu effect on RegIII gamma dose intervention group and related factors are most significant. In summary, lactic acid bacteria or its components of its structure and metabolites is not secreted can cause MyD88 to a certain extent, the increase in IL-23 and IL-22 and promote RegIII gamma increase. And lactic acid bacteria colonized with helps to reduce the inflammatory response, restore the immune balance to relieve enteritis effect. Further study showed that the lactic acid bacteria relief mechanism of IBD may be related to the decrease of RegIII gamma abnormal expression.

【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.3

【共引文献】