本文選題：旋毛蟲(chóng) + 腸道菌群��； 參考：《吉林農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：旋毛蟲(chóng)(Trichinella spiralis)屬于線蟲(chóng)動(dòng)物門(mén),無(wú)尾感器綱(Aphasmida)或有腺綱(Adenophorea),刺嘴目(Enoplida)。旋毛蟲(chóng)病是一種呈全球性分布的對(duì)于人類健康和畜牧業(yè)生產(chǎn)具有嚴(yán)重危害的人獸共患病。目前,旋毛蟲(chóng)相關(guān)研究已經(jīng)不僅僅局限于旋毛蟲(chóng)對(duì)宿主的致病作用和預(yù)防與治療方法,有關(guān)旋毛蟲(chóng)在免疫調(diào)節(jié)機(jī)制尤其是免疫逃避等倍受關(guān)注。近年來(lái),已有許多研究注意到腸道菌群在機(jī)體免疫、營(yíng)養(yǎng)代謝及疾病(如代謝綜合征,結(jié)直腸癌,肝癌,糖尿病等)的發(fā)生發(fā)展中的變化及作用,而關(guān)于寄生蟲(chóng)感染過(guò)程中腸道菌群變化相關(guān)研究相對(duì)滯后。腸道菌群具有調(diào)節(jié)非特異性免疫與特異性免疫的作用。本研究以旋毛蟲(chóng)感染過(guò)程中小鼠腸道菌群的變化為著眼點(diǎn),應(yīng)用Illumina高通量測(cè)序方法探究旋毛蟲(chóng)感染前后及感染不同時(shí)期小鼠腸道菌群變化情況,并應(yīng)用流式細(xì)胞術(shù)及ELISA等檢測(cè)方法研究小鼠免疫相關(guān)指標(biāo)的變化情況,以期為腸道菌群與旋毛蟲(chóng)免疫調(diào)節(jié)研究奠定基礎(chǔ),同時(shí)也為其他線蟲(chóng)感染腸道菌群與免疫調(diào)節(jié)研究提供參考。主要研究?jī)?nèi)容及結(jié)果如下:(1)旋毛蟲(chóng)感染對(duì)BALB/c小鼠腸道菌群的影響:將24只雌性SPF級(jí)BALB/c小鼠平均分為兩組,每組12只,一組為旋毛蟲(chóng)感染組,另一組為對(duì)照組。給旋毛蟲(chóng)組小鼠每只灌服旋毛蟲(chóng)肌幼蟲(chóng)300條,給對(duì)照組小鼠每只灌服等體積PBS。分別于感染后7天、14天、21天和28天無(wú)菌條件下采集小鼠新鮮糞便。提取糞便細(xì)菌基因組總DNA,然后對(duì)總DNA進(jìn)行16SrRNA基因序列Vl-V3高變區(qū)PCR擴(kuò)增產(chǎn)物進(jìn)行定量、混勻與Illumina高通量測(cè)序。測(cè)序分析結(jié)果表明旋毛蟲(chóng)感染7天、14天、21天、28天時(shí)小鼠腸道菌群OTU均高于對(duì)照組,所有序列屬于以下四個(gè)菌門(mén),分別為:擬桿菌門(mén)(Bacteroidetes)、硬壁菌門(mén)(Firmicutes)、形菌門(mén)(Proteobacteria)和脫鐵桿菌門(mén)(Deferribacteres),所占比例達(dá)99%以上。感染7天、14天、21天、28天時(shí)實(shí)驗(yàn)組小鼠腸道擬桿菌門(mén)細(xì)菌數(shù)量均高于對(duì)照組,且7天時(shí)兩組差異極顯著(P0.01);感染7天、14天、21天時(shí)實(shí)驗(yàn)組小鼠腸道硬壁菌門(mén)細(xì)菌數(shù)量均低于對(duì)照組,且7天時(shí)兩組差異極顯著(P0.01);感染28天時(shí)硬壁菌門(mén)細(xì)菌數(shù)量略高于對(duì)照組,差異不顯著(P0.05)。感染7天、14天、21天、28天時(shí)小鼠腸道變形桿菌門(mén)細(xì)菌數(shù)量均低于對(duì)照組,差異顯著(P0.05)。感染7天時(shí)小鼠腸道脫鐵桿菌門(mén)細(xì)菌數(shù)量高于對(duì)照組,差異顯著(P0.05);感染14天、21天、28天時(shí)實(shí)驗(yàn)組小鼠腸道脫鐵桿菌門(mén)細(xì)菌數(shù)量均低于對(duì)照組,其中感染28天時(shí)兩組差異顯著(P0.05)。感染旋毛蟲(chóng)后小鼠腸道菌群豐度及多樣性升高;腸道菌群變化逐漸趨于平緩。(2)菌群變化對(duì)感染旋毛蟲(chóng)小鼠免疫水平的影響:用流式細(xì)胞術(shù)檢測(cè)小鼠感染旋毛蟲(chóng)前后和感染不同時(shí)期的腸系膜淋巴結(jié)(MLN)Foxp3+Treg數(shù)量的變化情況、Th2型細(xì)胞因子IL-10及Th17型細(xì)胞因子IL-17表達(dá)量的變化情況;并采用間接ELISA法檢測(cè)小鼠感染旋毛蟲(chóng)前后和感染不同時(shí)期血清IFN-γ,IL-4,IL-22,IL-17表達(dá)量變化情況分析旋毛蟲(chóng)感染對(duì)小鼠免疫的影響。結(jié)果顯示在旋毛蟲(chóng)感染過(guò)程中Th2型細(xì)胞因子分泌量始終高于對(duì)照組,呈上升趨勢(shì);而Th1型細(xì)胞因子分泌量始終低于對(duì)照組,呈下降趨勢(shì);Treg數(shù)量始終高于對(duì)照組,也呈上升趨勢(shì);Th17型細(xì)胞因子在旋毛蟲(chóng)感染前期分泌量高于對(duì)照組,感染后期基本與對(duì)照組持平。Th1型、Th2型、Th17型細(xì)胞因子水平隨著旋毛蟲(chóng)感染時(shí)間的延長(zhǎng),變化趨勢(shì)逐漸趨于平緩。因此可以推斷在旋毛蟲(chóng)感染過(guò)程中腸道菌群的變化對(duì)小鼠免疫應(yīng)答起到了積極的推動(dòng)作用。腸道菌群與各種細(xì)胞因子協(xié)同調(diào)節(jié)腸道,使其盡快恢復(fù)到相對(duì)穩(wěn)定狀態(tài),以達(dá)到腸道菌群和旋毛蟲(chóng)的共生。以上結(jié)果為腸道菌群與線蟲(chóng)感染免疫之間的關(guān)系研究提供參考,同時(shí)為腸道寄生線蟲(chóng)病的防治及腸道炎性疾病生物學(xué)療法提供新的思路。
[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.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.7

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