旋毛虫感染小鼠肠道菌群变化与免疫应答关系初步研究

发布时间：2018-06-22 18:48

本文选题：旋毛虫 + 肠道菌群　；参考：《吉林农业大学》2017年硕士论文

【摘要】：旋毛虫(Trichinella spiralis)属于线虫动物门,无尾感器纲(Aphasmida)或有腺纲(Adenophorea),刺嘴目(Enoplida)。旋毛虫病是一种呈全球性分布的对于人类健康和畜牧业生产具有严重危害的人兽共患病。目前,旋毛虫相关研究已经不仅仅局限于旋毛虫对宿主的致病作用和预防与治疗方法,有关旋毛虫在免疫调节机制尤其是免疫逃避等倍受关注。近年来,已有许多研究注意到肠道菌群在机体免疫、营养代谢及疾病(如代谢综合征,结直肠癌,肝癌,糖尿病等)的发生发展中的变化及作用,而关于寄生虫感染过程中肠道菌群变化相关研究相对滞后。肠道菌群具有调节非特异性免疫与特异性免疫的作用。本研究以旋毛虫感染过程中小鼠肠道菌群的变化为着眼点,应用Illumina高通量测序方法探究旋毛虫感染前后及感染不同时期小鼠肠道菌群变化情况,并应用流式细胞术及ELISA等检测方法研究小鼠免疫相关指标的变化情况,以期为肠道菌群与旋毛虫免疫调节研究奠定基础,同时也为其他线虫感染肠道菌群与免疫调节研究提供参考。主要研究内容及结果如下:(1)旋毛虫感染对BALB/c小鼠肠道菌群的影响:将24只雌性SPF级BALB/c小鼠平均分为两组,每组12只,一组为旋毛虫感染组,另一组为对照组。给旋毛虫组小鼠每只灌服旋毛虫肌幼虫300条,给对照组小鼠每只灌服等体积PBS。分别于感染后7天、14天、21天和28天无菌条件下采集小鼠新鲜粪便。提取粪便细菌基因组总DNA,然后对总DNA进行16SrRNA基因序列Vl-V3高变区PCR扩增产物进行定量、混匀与Illumina高通量测序。测序分析结果表明旋毛虫感染7天、14天、21天、28天时小鼠肠道菌群OTU均高于对照组,所有序列属于以下四个菌门,分别为:拟杆菌门(Bacteroidetes)、硬壁菌门(Firmicutes)、形菌门(Proteobacteria)和脱铁杆菌门(Deferribacteres),所占比例达99%以上。感染7天、14天、21天、28天时实验组小鼠肠道拟杆菌门细菌数量均高于对照组,且7天时两组差异极显著(P0.01);感染7天、14天、21天时实验组小鼠肠道硬壁菌门细菌数量均低于对照组,且7天时两组差异极显著(P0.01);感染28天时硬壁菌门细菌数量略高于对照组,差异不显著(P0.05)。感染7天、14天、21天、28天时小鼠肠道变形杆菌门细菌数量均低于对照组,差异显著(P0.05)。感染7天时小鼠肠道脱铁杆菌门细菌数量高于对照组,差异显著(P0.05);感染14天、21天、28天时实验组小鼠肠道脱铁杆菌门细菌数量均低于对照组,其中感染28天时两组差异显著(P0.05)。感染旋毛虫后小鼠肠道菌群丰度及多样性升高;肠道菌群变化逐渐趋于平缓。(2)菌群变化对感染旋毛虫小鼠免疫水平的影响:用流式细胞术检测小鼠感染旋毛虫前后和感染不同时期的肠系膜淋巴结(MLN)Foxp3+Treg数量的变化情况、Th2型细胞因子IL-10及Th17型细胞因子IL-17表达量的变化情况;并采用间接ELISA法检测小鼠感染旋毛虫前后和感染不同时期血清IFN-γ,IL-4,IL-22,IL-17表达量变化情况分析旋毛虫感染对小鼠免疫的影响。结果显示在旋毛虫感染过程中Th2型细胞因子分泌量始终高于对照组,呈上升趋势;而Th1型细胞因子分泌量始终低于对照组,呈下降趋势;Treg数量始终高于对照组,也呈上升趋势;Th17型细胞因子在旋毛虫感染前期分泌量高于对照组,感染后期基本与对照组持平。Th1型、Th2型、Th17型细胞因子水平随着旋毛虫感染时间的延长,变化趋势逐渐趋于平缓。因此可以推断在旋毛虫感染过程中肠道菌群的变化对小鼠免疫应答起到了积极的推动作用。肠道菌群与各种细胞因子协同调节肠道,使其尽快恢复到相对稳定状态,以达到肠道菌群和旋毛虫的共生。以上结果为肠道菌群与线虫感染免疫之间的关系研究提供参考,同时为肠道寄生线虫病的防治及肠道炎性疾病生物学疗法提供新的思路。
[Abstract]:Trichinella (Trichinella spiralis) belongs to the nematode gate, Aphasmida or Adenophorea, and Enoplida. Trichinosis is a worldwide zoonosis which is seriously harmful to human health and animal production. At present, the research of Trichinella spiralis has not only been limited to spin hair. In recent years, many studies have paid attention to the changes and development of intestinal flora in the development of body immunity, nutrition metabolism and diseases (such as metabolic syndrome, colorectal cancer, liver cancer, diabetes and so on). Research on intestinal microflora changes in the process of parasitic infection is relatively lagging. Intestinal microflora has the role of regulating non specific immunity and specific immunity. This study focuses on the changes in the intestinal flora of Trichinella Trichinella in the process of Trichinella infection, and the application of Illumina high throughput sequencing to explore the infection and infection of Trichinella Trichinella The changes of intestinal microflora in different periods of mice were studied, and the changes of immune related indexes in mice were studied by flow cytometry and ELISA, in order to lay the foundation for the immunoregulation of intestinal flora and Trichinella, and also to provide reference for the study of intestinal flora and immunoregulation of other nematodes. The results are as follows: (1) the effect of Trichinella infection on the intestinal flora of BALB/c mice: 24 female SPF grade BALB/c mice were divided into two groups, each group was a Trichinella infection group and the other group was a control group. Each group of Trichinella Trichinella mice was given 300 Trichinella larvae each, and the same volume of PBS. in the control group was respectively infected. After 7 days, 14 days, 14 days, 21 days and 28 days, the fresh feces of mice were collected. The total genomic DNA of the fecal bacterial genome was extracted, then the PCR amplification products of the Vl-V3 high variable region of the total 16SrRNA gene sequence were quantified and mixed with Illumina high throughput sequencing. The results of sequencing analysis showed that the intestinal flora O was 7 days, 14 days, 21 days and 28 days. TU was higher than that of the control group, and all the sequence belonged to the following four bacteria doors, respectively: Bacteroidetes, Firmicutes, Proteobacteria and Deferribacteres, which accounted for more than 99%. Infection 7 days, 14 days, 21 days, and 28 days, the number of bacteria in the intestinal bacteriobacillus in experimental group was higher than the control. The difference between the two groups was very significant (P0.01) at 7 days, and the number of bacteria in the intestinal hard wall bacteria in the experimental group was lower than the control group at 7 days, 14 days and 21 days, and the two groups were significantly different at 7 days (P0.01), and the number of bacteria in the hard wall bacteria was slightly higher than that of the control group at the 28 day of infection (P0.05). Infection 7 days, 14 days, 21 days, and 28 days of mice intestinal change. The number of bacteria in the bacilli gate was lower than that of the control group (P0.05). The number of bacteria in intestinal defermebacterium gate in mice was higher than that of the control group (P0.05) at 7 days. The number of bacteria in the intestinal deferring bacilli in the experimental group was lower than that in the control group at 14 days, 21 days and 28 days, and the difference between the two groups was significant (P0.05) when the infection was 28 days. The abundance and diversity of intestinal microflora in mice after caterpillar increased, and the changes of intestinal flora gradually tended to slow. (2) the effect of change on the immune level of the mice infected with Trichinella Trichinella infection: the change of the number of mesenteric lymph nodes (MLN) Foxp3+Treg in the mice infected with Trichinella Trichinella and the different stages of infection by flow cytometry, and the Th2 cytokine I The change of the expression of L-10 and Th17 type cytokine IL-17, and the changes of the expression of IFN- gamma, IL-4, IL-22 and IL-17 in the serum of Trichinella Trichinella infected by Trichinella Trichinella and the infection in different periods by indirect ELISA were used to analyze the effect of Trichinella infection on the immunity of mice. The results showed that the secretion of Th2 type cytokines was beginning in the process of the infection of the Trichinella. The level of Th1 cytokine secretion was always lower than that of the control group, and the number of Treg was always higher than that of the control group. The secretion of Th17 type cytokines in the early stage of Trichinella Trichinella infection was higher than that of the control group, and the later stage of infection was basically.Th1, Th2 and Th17 cytokine levels. With the prolongation of the time of Trichinella Trichinella infection, the change trend gradually tends to be slow. Therefore, it can be concluded that the changes of intestinal flora in the process of Trichinella infection have played an active role in promoting the immune response in mice. Intestinal flora and various cytokines regulate the intestinal tract, so that the intestinal microflora can be restored to the relatively stable state as soon as possible to achieve the intestinal flora. The above results provide a reference for the study of the relationship between intestinal flora and nematode infection immunity, and provide new ideas for the prevention and treatment of intestinal parasitic nematode and the biological therapy of intestinal inflammatory diseases.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.7

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