嗜酸乳杆菌胞外多糖对TGEV感染ST细胞TLR3及部分细胞因子表达的影响

发布时间：2018-04-30 19:35

本文选题：嗜酸乳杆菌胞外多糖 + 猪传染性胃肠炎病毒　；参考：《东北农业大学》2015年硕士论文

【摘要】：益生菌以其天然、安全、无毒、无残留等特点在畜牧业行业越来越受到广泛地关注。目前,有关益生菌在抑菌、调节免疫等方面的研究有很多报道,但是对于益生菌抗病毒作用的报道比较有限。猪传染性胃肠炎病毒(TGEV)可引起猪传染性胃肠炎,本病严重威胁养殖业的发展,对该病的防制尤为重要。因此,本研究选择一株具有抗病毒活性的嗜酸乳杆菌,根据MTT法检测确定其代谢产物中的胞外多糖(EPS)具有抑制TGEV感染猪睾丸细胞(ST)的作用,据此开展了嗜酸乳杆菌胞外多糖对TGEV感染ST细胞的TLR3、细胞因子IFN-γ、IL-6、IL-8以及细胞信号通路核转录因子NF-κB表达水平影响的研究,探索益生菌胞外多糖抗病毒感染的细胞机制。试验将铺满单层的ST细胞进行分组处理,分为四组:正常细胞对照组(control组)、TGEV感染组(TGEV组)、胞外多糖预处理感染TGEV组(EPS+TGEV组)和胞外多糖组(EPS组)。四组处理方案分别在2h、4h、6h、12h、24h时采集细胞上清和细胞样品。采用实时荧光定量PCR方法检测了各组细胞样品TLR3、IFN-γ、IL-6、IL-8 m RNA表达水平,该方法是根据Gen Bank中登录的TLR3、IFN-γ、IL-6、IL-8和管家基因(β-actin)序列以及参考文献中的引物序列设计并合成荧光引物,建立实时荧光定量PCR检测方法;采用ELISA方法测定了各组细胞上清IFN-γ、IL-6、IL-8蛋白表达水平;采用Western blot方法检测了细胞信号通路核转录因子NF-κB表达水平。试验结果数据经统计分析表明,EPS组、TGEV组和EPS+TGEV组TLR3、IFN-γ、IL-6、IL-8 m RNA表达量较正常细胞对照组比都相对增加,随着时间的延长,均呈现出先快速增加后减少回归到正常水平的趋势。6h后大多数处理组TLR3、IFN-γ、IL-6、IL-8 m RNA表达量趋于正常水平,与正常细胞对照组比无显著变化。EPS+TGEV组在感染2h时IL-8 m RNA表达量达到最大值,4h时IL-6、IFN-γm RNA表达量达到最大值,且均出现了叠加效应,相比正常细胞对照组差异极显著。ELISA检测结果与荧光定量PCR检测结果整体趋势是相同的。Western blot研究结果显示,嗜酸乳杆菌胞外多糖和TGEV都能刺激ST细胞信号通路核转录因子NF-κB蛋白的表达。EPS+TGEV组、TGEV组、EPS组的NF-κB核内表达含量均高于正常细胞对照组,且EPS+TGEV组的NF-κB核内表达含量高于TGEV组、EPS组。以上研究结果表明,嗜酸乳杆菌胞外多糖和TGEV均可以诱导细胞表面受体TLR3的表达从而启动下游NF-κB途径并诱发细胞因子IFN-γ、IL-6、IL-8的分泌,刺激细胞的先天性免疫反应,增强细胞对病毒的抵抗力。嗜酸乳杆菌胞外多糖预处理再感染TGEV也产生同样的效果,并且TLR3、IFN-γ、IL-6、IL-8的表达量出现了叠加效应。该研究为益生菌胞外多糖在诱导细胞因子表达,参与抵抗病毒感染方面的益生菌抗病毒作用机制研究提供了新的依据。
[Abstract]:Probiotics have attracted more and more attention in animal husbandry industry because of its natural, safe, non-toxic and non-residual characteristics. At present, there are a lot of reports about probiotics in bacteriostasis and immune regulation, but the antiviral effects of probiotics are limited. Infectious gastroenteritis virus (TGEV) can cause infectious gastroenteritis in pigs, which is a serious threat to the development of aquaculture, and is particularly important for the prevention and control of the disease. Therefore, a strain of Lactobacillus acidophilus with antiviral activity was selected in this study. It was determined by MTT assay that the extracellular polysaccharide (EPS) in its metabolites could inhibit TGEV infection in porcine testicular cells. The effects of EPS from Lactobacillus acidophilus on the expression of TLR3, IFN- 纬 -IL-6IL-8 and NF- 魏 B in St cells infected with TGEV were studied, and the cellular mechanism of anti-viral infection of EPS from probiotics was explored. St cells covered with monolayer were divided into four groups: normal cell control group (control group), control group (TGEV infected group), exopolysaccharide preconditioning group (TGEV group) and exopolysaccharide group (EPS group). Cell supernatants and cell samples were collected at 2 h ~ 4 h ~ 6 h ~ 12 h ~ 24 h. Real-time fluorescence quantitative PCR was used to detect the expression of IL-6mIL-8 m RNA in TLR3hIFN- 纬 -tl. The primers were designed and synthesized according to the sequences of IL-6IL-8 and 尾 -actinin of TLR3IFN- 纬 IFN- 纬, and the primer sequences of reference literature. A real-time fluorescent quantitative PCR assay was established, and the expression of IFN- 纬 -hIL-6 IL-8 in the supernatant of each group was determined by ELISA method, and the expression of nuclear transcription factor NF- 魏 B in the cell signaling pathway was detected by Western blot method. The statistical analysis showed that the expression of IL-6 IL-8 m RNA in TGEV group and EPS TGEV group was higher than that in normal control group, and the expression of IL-8 m RNA in TLR3, IFN- 纬, IFN- 纬, and TLR3IFN- 纬 was higher than that in normal control group, and the expression of IL-8 m in TGEV group and EPS TGEV group was increased. After 6 h, the expression of IL-8 m RNA in TLR3IFN- 纬 ~ (-) ~ (-) ~ (-) ~ (-) IL-6 ~ (-) was increased to normal level in most of the treatment groups. In EPS TGEV group, the expression of IL-8 m RNA reached the maximum at 2 h after infection and the expression of IL-6 + IFN- 纬 m RNA reached the maximum at 4 h after infection, and there was a superposition effect on the expression of IL-6 + IFN- 纬 m RNA in EPS TGEV group. The results of Elisa and fluorescence quantitative PCR showed that the overall trend was the same as that of the control group. Lactobacillus acidophilus exopolysaccharide and TGEV could stimulate the expression of NF- 魏 B protein in St cell signaling pathway. The expression of NF- 魏 B in EPS TGEV group was higher than that in normal cell control group. The expression of NF- 魏 B in EPS TGEV group was higher than that in TGEV group. These results indicate that both Lactic acidophilus extracellular polysaccharides and TGEV can induce the expression of TLR3 on the cell surface, thus initiate the downstream NF- 魏 B pathway, induce the secretion of cytokine IFN- 纬 -IL-6 and IL-8, and stimulate the innate immune response of the cells. Enhance cell resistance to viruses. The same effect was also found in the pretreatment and reinfection of TGEV by Lactobacillus acidophilus extracellular polysaccharides, and the superposition effect was found in the expression of IL-6 and IL-8 in TLR3 IFN- 纬 -IFN- 纬. This study provides a new basis for the study of the antiviral mechanism of probiotic extracellular polysaccharides in inducing cytokine expression and participating in the resistance to viral infection.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

【参考文献】