本文選題：嗜酸乳桿菌胞外多糖 + 豬傳染性胃腸炎病毒　； 參考：《東北農(nóng)業(yè)大學》2015年碩士論文

【摘要】：益生菌以其天然、安全、無毒、無殘留等特點在畜牧業(yè)行業(yè)越來越受到廣泛地關注。目前,有關益生菌在抑菌、調(diào)節(jié)免疫等方面的研究有很多報道,但是對于益生菌抗病毒作用的報道比較有限。豬傳染性胃腸炎病毒(TGEV)可引起豬傳染性胃腸炎,本病嚴重威脅養(yǎng)殖業(yè)的發(fā)展,對該病的防制尤為重要。因此,本研究選擇一株具有抗病毒活性的嗜酸乳桿菌,根據(jù)MTT法檢測確定其代謝產(chǎn)物中的胞外多糖(EPS)具有抑制TGEV感染豬睪丸細胞(ST)的作用,據(jù)此開展了嗜酸乳桿菌胞外多糖對TGEV感染ST細胞的TLR3、細胞因子IFN-γ、IL-6、IL-8以及細胞信號通路核轉(zhuǎn)錄因子NF-κB表達水平影響的研究,探索益生菌胞外多糖抗病毒感染的細胞機制。試驗將鋪滿單層的ST細胞進行分組處理,分為四組:正常細胞對照組(control組)、TGEV感染組(TGEV組)、胞外多糖預處理感染TGEV組(EPS+TGEV組)和胞外多糖組(EPS組)。四組處理方案分別在2h、4h、6h、12h、24h時采集細胞上清和細胞樣品。采用實時熒光定量PCR方法檢測了各組細胞樣品TLR3、IFN-γ、IL-6、IL-8 m RNA表達水平,該方法是根據(jù)Gen Bank中登錄的TLR3、IFN-γ、IL-6、IL-8和管家基因(β-actin)序列以及參考文獻中的引物序列設計并合成熒光引物,建立實時熒光定量PCR檢測方法;采用ELISA方法測定了各組細胞上清IFN-γ、IL-6、IL-8蛋白表達水平;采用Western blot方法檢測了細胞信號通路核轉(zhuǎn)錄因子NF-κB表達水平。試驗結(jié)果數(shù)據(jù)經(jīng)統(tǒng)計分析表明,EPS組、TGEV組和EPS+TGEV組TLR3、IFN-γ、IL-6、IL-8 m RNA表達量較正常細胞對照組比都相對增加,隨著時間的延長,均呈現(xiàn)出先快速增加后減少回歸到正常水平的趨勢。6h后大多數(shù)處理組TLR3、IFN-γ、IL-6、IL-8 m RNA表達量趨于正常水平,與正常細胞對照組比無顯著變化。EPS+TGEV組在感染2h時IL-8 m RNA表達量達到最大值,4h時IL-6、IFN-γm RNA表達量達到最大值,且均出現(xiàn)了疊加效應,相比正常細胞對照組差異極顯著。ELISA檢測結(jié)果與熒光定量PCR檢測結(jié)果整體趨勢是相同的。Western blot研究結(jié)果顯示,嗜酸乳桿菌胞外多糖和TGEV都能刺激ST細胞信號通路核轉(zhuǎn)錄因子NF-κB蛋白的表達。EPS+TGEV組、TGEV組、EPS組的NF-κB核內(nèi)表達含量均高于正常細胞對照組,且EPS+TGEV組的NF-κB核內(nèi)表達含量高于TGEV組、EPS組。以上研究結(jié)果表明,嗜酸乳桿菌胞外多糖和TGEV均可以誘導細胞表面受體TLR3的表達從而啟動下游NF-κB途徑并誘發(fā)細胞因子IFN-γ、IL-6、IL-8的分泌,刺激細胞的先天性免疫反應,增強細胞對病毒的抵抗力。嗜酸乳桿菌胞外多糖預處理再感染TGEV也產(chǎn)生同樣的效果,并且TLR3、IFN-γ、IL-6、IL-8的表達量出現(xiàn)了疊加效應。該研究為益生菌胞外多糖在誘導細胞因子表達,參與抵抗病毒感染方面的益生菌抗病毒作用機制研究提供了新的依據(jù)。
[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- 緯 -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- 緯 -tl. The primers were designed and synthesized according to the sequences of IL-6IL-8 and 尾 -actinin of TLR3IFN- 緯 IFN- 緯, and the primer sequences of reference literature. A real-time fluorescent quantitative PCR assay was established, and the expression of IFN- 緯 -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- 緯, IFN- 緯, and TLR3IFN- 緯 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- 緯 ~ (-) ~ (-) ~ (-) ~ (-) 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- 緯 m RNA reached the maximum at 4 h after infection, and there was a superposition effect on the expression of IL-6 + IFN- 緯 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- 緯 -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- 緯 -IFN- 緯. 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.
【學位授予單位】：東北農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.65

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