旋毛虫感染小鼠肠道免疫应答及菌群变化规律的研究

发布时间：2018-10-18 08:06

【摘要】：胃肠道中含有大量的微生物,其中包括细菌、真菌、酵母和寄生虫等。肠道正常菌群参与机体的生理、生化及病理过程,与机体形成互惠互利、相互协调又相互制约的动态平衡关系,同时肠道正常菌群在肠黏膜表面构成一个重要的免疫屏障,可有效抵御外来病原体的入侵和繁殖等。肠道中的抗菌肽或蛋白也是宿主重要的防御性分子,在防止宿主发生感染和维持内环境稳定方面发挥着积极作用,其中包括Mucin、Intelectin、溶菌酶和防御素等。自噬(autophage)是继凋亡(apoptosis)后,当前生命科学最热的研究领域,是广泛存在于真核细胞内的一种溶酶体依赖性的降解途径,在机体生长、发育和疾病过程中发挥着重要作用,对天然免疫和获得性免疫具有一定的调节作用。旋毛形线虫(Trichinella spiralis,旋毛虫)感染为胃肠道紊乱病理生理学的研究提供了一个很好的实验模型,在过去的十年中,人们利用旋毛虫感染大鼠或小鼠模型来研究免疫和肠道生理系统的相互关系以及相关机理等。目前,人们着重于研究细菌和肠黏膜上皮细胞发生相互作用后肠道抗菌肽表达水平的变化,而对于寄生虫感染后抗菌肽表达水平的变化,是否引起自噬现象的发生,以及肠道菌群变化的研究较少,此外,这三者之间的相互关系等尚不清楚。因此,为了探讨旋毛虫ISS534感染BALB/c小鼠的肠道免疫应答机制,本研究对旋毛虫感染BALB/c小鼠后肠道内细胞因子、抗菌肽、自噬以及菌群变化等相关宿主防御反应进行检测。 6-8周龄BALB/c小鼠经口感染300尾旋毛虫ISS534幼虫／只,在感染7d和14d后断颈处死小鼠,采集空肠和结肠进行体外组织培养,培养18h后收集肠组织培养上清,通过ELISA方法对IL-10、IL-12、IL-6、IL-17、IFN-y和TNF-a等细胞因子的表达量进行检测,进一步验证了旋毛虫感染小鼠后机体的免疫反应是体液免疫和细胞免疫协同作用结果,其中以Th2型免疫反应为主。之后,采集小鼠空肠和结肠进行石蜡切片,经HE染色后观察,可见旋毛虫ISS534感染BALB/c小鼠空肠中杯状细胞数量增加；Real-time PCR检测结果表明,与对照组相比,由杯状细胞分泌的Intelectin和Mucinl,潘氏细胞分泌的溶菌酶和鼠防御素-1表达量增加,说明抗菌肽在宿主感染旋毛虫后引发的免疫应答中发挥着积极作用。此外,利用Western blot方法对自噬体膜标志性蛋白LC3B表达水平进行检测,结果发现旋毛虫ISS534感染BALB/c小鼠空肠和结肠中LC3B表达量均增高,尤其是感染后结肠中LC3B表达量与对照组相比显著增加,这说明在寄生虫感染情况下,自噬很可能也参与了机体的免疫防御反应。根据不同细菌的16S rRNA序列设计特异性引物,构建阳性质粒作为标准品,应用Real-time PCR方法对旋毛虫ISS534感染BALB/c小鼠肠道中的乳酸杆菌属、双歧杆菌属、肠球菌属和梭菌属细菌进行定量分析,比较旋毛虫感染小鼠与正常对照之间肠道菌群差异。该试验结果表明,旋毛虫ISS534感染BALB/c小鼠后可引起肠道菌群失调。
[Abstract]:The gastrointestinal tract contains a large number of microorganisms, including bacteria, fungi, yeast, and parasites. the intestinal normal bacteria group participates in the physiological, biochemical and pathological processes of the organism and forms a dynamic balance relationship with the body to form mutual benefit, mutual coordination and mutual restriction, and meanwhile, the intestinal normal bacteria group forms an important immune barrier on the surface of the intestinal mucosa, can effectively resist invasion and propagation of foreign pathogens and the like. The antibacterial peptide or protein in the intestinal tract is also an important defensive molecule of the host, and plays an active role in preventing the host from infecting and maintaining homeostasis, including Mucin, Inelectroin, Lysozyme and Defensin. Since autophagy is the most hot research field of life science after apoptosis, it is a kind of lysosome-dependent degradation pathway widely present in eukaryotic cells, which plays an important role in organism growth, development and disease process. It has a certain regulating effect on innate immunity and immunity. Trichinella spiralis (Trichinella spiralis, Trichinella spiralis) infection provides a good experimental model in the study of gastrointestinal disorder pathophysiology, In the past ten years, people use Trichinella spiralis infection rats or mouse models to study the correlation between the immune and intestinal physiological systems and the correlation Mechanism and so on. At present, people focus on studying the change of the expression level of antibacterial peptide after the interaction of bacteria and intestinal mucosa epithelial cells, and whether the change of the expression level of antibacterial peptide after the parasite infection causes autophagy, and little research on intestinal flora changes. In addition, the relations among the three are not yet Therefore, in order to explore the mechanism of intestinal immune response in BALB/ c mice infected with Trichinella spiralis ISS534, this study has been used to investigate the immune response to cytokines, antibacterial peptides, autophagy and changes of bacteria group in BALB/ c mice infected with trichinella spiralis. 6-8 week old BALB/ c mice were infected with 300 tail trichinella spiralis ISS534 larvae/ only, the mice were sacrificed after 7 days after infection and 14 days after infection, the samples were collected in vitro for culture in vitro and cultured in vitro for 18h, IL-10, IL-12, IL-12, IL-12 and IL-12 were collected by ELISA. 6. The expression of cytokines such as IL-17, IFN-y and TNF-a were detected, and the immune response of the mice after Trichinella spiralis infection was further verified to be the result of humoral immunity and cellular immunity. After HE staining, the number of goblet cells was increased in BALB/ c mice infected with Trichinella spiralis ISS534, and the results of Real-time PCR showed that, compared with the control group, Inelectroin, which was secreted by cup-shaped cells, was observed after HE staining. and the expression amount of the lysozyme and the mouse defensin-1 secreted by the Mucinl and Panshi cells is increased, and the immune response induced by the antibacterial peptide after the host infection is infected In addition, the expression level of L3B was detected by Western blot, and the expression level of L3B in the jejunal and colon of BALB/ c mice infected with Trichinella spiralis was increased, especially the expression of L3B in the colon after infection and control. This indicates that autophagy is likely to be involved in the body as compared to a significant increase The specific primers were designed according to 16S rRNA sequence of different bacteria to construct a positive plasmid as a standard. Quantitative analysis of bacteria to compare mice infected with Trichinella spiralis with normal control The results showed that the ISS534 infected with Trichinella spiralis could lead to BALB/ c mice.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392

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