猪源益生菌对宿主肠道病毒的抗病毒作用研究
发布时间：2018-08-08 20:16
【摘要】：益生菌的应用已持续数十年,虽然其益生作用机理尚不清楚,但在临床上对消化道疾病的显著疗效已经得到充分的证实,对机体免疫功能的保护和增强作用愈加受到关注。一般认为益生菌在临床治疗肠道感染性腹泻的分子和细胞机制主要涉及三个方面:(1)益生菌可产生抗菌物质并竞争性与病原性微生物黏附于肠上皮细胞来阻止病原体在肠道的黏附和定植;(2)益生菌可提高病毒感染时肠上皮细胞的存活率和保护肠道的正常生理结构并刺激保护性免疫反应来调节机体免疫;(3)益生菌可通过调节肠道微生态平衡来保护和抵御病原微生物的侵袭。本研究主要从分子水平、细胞水平以及动物体内试验三个方面阐述猪肠道益生乳酸菌对传染性胃肠炎病毒(TGEV)和猪流行性腹泻病毒(PEDV)的抑制作用和免疫调节作用,获得了以下结果:(1)分离筛选出在猪睾丸细胞(ST细胞)上对TGEV具有抑制作用的3株猪源肠道益生菌Y091231、Y091221和Y224031,经16S rDNA鉴定分别为唾液乳杆菌(Lactobacillus saliarius),约氏乳杆菌(Lactobacillus johnsonii)和粪肠球菌(Enterococcus faecalis)。(2)筛选菌对感染TGEV的ST细胞具有保护作用。3株益生菌及其代谢产物接入顺序不同,对病毒的抑制率也不同,Y091231菌悬液在预先接入的情况下可以达到最大抑制率(65%),而Y091221和Y224031则在与病毒共同接入时表现出较高的抑制率。通过半定量RT-PCR检测结果可知,3株益生菌及其代谢产物均可以降低TGEV在ST细胞内的增殖,Y091231菌悬液在各处理组中具有稳定的较高的病毒抑制力。益生菌在体外抗TGEV作用中,可以通过上调IL-8和IL-12、下调IL-4和INF-α这4种细胞因子在细胞培养上清中的表达量,从而刺激细胞免疫,起到抵御病毒入侵的作用。(3)分离鉴定PEDV-WN株,构建动物模型。从临床病料中分离得到PEDV,然后对毒株进行RT-PCR鉴定及M、S基因序列分析,与CV777株对比分析,PEDV-WN株的M基因具有高度保守性,可以作为检测PEDV的靶基因;S基因变异较大,S蛋白内分的核心和表位(CEO,499-638aa)共有9个氨基酸位点发生了突变。将PEDV-WN株回归试验动物,成功构建了典型PEDV感染动物模型。(4)唾液乳杆菌(Lactobacillus saliarius)对PEDV-WN株具有抑制作用,对病毒感染的仔猪具有保护作用。该菌株的预防性干预和治疗性干预均能提高仔猪存活率,提高仔猪生长性能,减轻病毒对肠道组织的病理损伤,并能降低病毒在机体内的增殖。益生菌可以调节机体IL-4、IL-8、IL-12、INF-α4种细胞因子和MHCⅡ的表达量,从而刺激机体的细胞免疫和体液免疫,起到抗病毒的作用。(5)PEDV感染严重破坏了正常的肠道菌群结构和菌种丰度,唾液乳杆菌的干预可以保护肠道微生态保持健康的平衡状态,并能迅速修复失调的肠道微生态。通过对粪便菌群的16S rDNA高通量测序的方法,对PEDV感染和益生菌干预的不同试验组的肠道菌群的变化规律和差异性进行了对比分析。
[Abstract]:The application of probiotics has lasted for decades. Although the mechanism of probiotics is not clear, the remarkable effect of probiotics on digestive tract diseases has been fully confirmed, and the protection and enhancement of immune function have been paid more and more attention. It is generally believed that the molecular and cellular mechanisms of probiotics in the treatment of intestinal infectious diarrhea are mainly related to three aspects: (1) probiotics can produce antimicrobial substances and be adhered to intestinal epithelial cells by competitive and pathogenic microorganisms to prevent pathogens. (2) probiotics can improve the survival rate of intestinal epithelial cells and protect the normal physiological structure of the intestine and stimulate the protective immune response to regulate the immune system; (3) probiotics can regulate the immune system by regulating the intestinal tract. Tao microecological balance to protect and protect against the invasion of pathogenic microorganisms. The inhibitory and immunomodulatory effects of porcine enteric probiotic lactic acid bacteria on infectious gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) were discussed from three aspects: molecular level, cell level and animal test in vivo. The following results were obtained: (1) three strains of porcine enteroprobiotics Y091231, Y091221 and Y224031 were isolated and screened to inhibit TGEV in porcine testicular cells (St cells). They were identified as Lactobacillus saliva (Lactobacillus saliarius), (Lactobacillus johnsonii) and fecal intestine by 16s rDNA, respectively. Streptococcus (Enterococcus faecalis). (_ 2) showed a protective effect on St cells infected with TGEV. 3 strains of probiotics and their metabolites were inserted in different sequences. The inhibition rate of Y091231 strain was also different, and that of Y091221 and Y224031 was higher than that of Y091221 and Y224031. The results of semi-quantitative RT-PCR showed that three strains of probiotics and their metabolites could reduce the proliferation of TGEV in St cells. Probiotics can up-regulate the expression of IL-8 and IL-12 and down-regulate the expression of IL-4 and INF- 伪 in the supernatant of cell culture, thus stimulating cellular immunity and resisting virus invasion. (3) isolation and identification of PEDV-WN strain. Build animal model. PEDVV was isolated from clinical materials. The M gene of PEDV-WN strain was highly conserved by RT-PCR identification and sequence analysis of MNs gene of PEDV-WN strain, and compared with CV777 strain, the M gene of PEDV-WN strain was highly conserved. A total of 9 amino acid loci were mutated at the core and epitope (CEO 499-638aa), which can be used as the target gene for PEDV detection. The typical animal model of PEDV infection was successfully constructed by regression of PEDV-WN strain. (4) Lactobacillus saliva (Lactobacillus saliarius) inhibited PEDV-WN strain and protected piglets infected by the virus. The preventive and therapeutic intervention of the strain can improve the survival rate of piglets, improve the growth performance of piglets, alleviate the pathological damage caused by the virus to the intestinal tissue, and reduce the proliferation of the virus in the body. Probiotics can regulate the expression of IL-4, IL-8, IL-12, INF- 伪 and MHC 鈪，
本文编号：2172933
[Abstract]:The application of probiotics has lasted for decades. Although the mechanism of probiotics is not clear, the remarkable effect of probiotics on digestive tract diseases has been fully confirmed, and the protection and enhancement of immune function have been paid more and more attention. It is generally believed that the molecular and cellular mechanisms of probiotics in the treatment of intestinal infectious diarrhea are mainly related to three aspects: (1) probiotics can produce antimicrobial substances and be adhered to intestinal epithelial cells by competitive and pathogenic microorganisms to prevent pathogens. (2) probiotics can improve the survival rate of intestinal epithelial cells and protect the normal physiological structure of the intestine and stimulate the protective immune response to regulate the immune system; (3) probiotics can regulate the immune system by regulating the intestinal tract. Tao microecological balance to protect and protect against the invasion of pathogenic microorganisms. The inhibitory and immunomodulatory effects of porcine enteric probiotic lactic acid bacteria on infectious gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) were discussed from three aspects: molecular level, cell level and animal test in vivo. The following results were obtained: (1) three strains of porcine enteroprobiotics Y091231, Y091221 and Y224031 were isolated and screened to inhibit TGEV in porcine testicular cells (St cells). They were identified as Lactobacillus saliva (Lactobacillus saliarius), (Lactobacillus johnsonii) and fecal intestine by 16s rDNA, respectively. Streptococcus (Enterococcus faecalis). (_ 2) showed a protective effect on St cells infected with TGEV. 3 strains of probiotics and their metabolites were inserted in different sequences. The inhibition rate of Y091231 strain was also different, and that of Y091221 and Y224031 was higher than that of Y091221 and Y224031. The results of semi-quantitative RT-PCR showed that three strains of probiotics and their metabolites could reduce the proliferation of TGEV in St cells. Probiotics can up-regulate the expression of IL-8 and IL-12 and down-regulate the expression of IL-4 and INF- 伪 in the supernatant of cell culture, thus stimulating cellular immunity and resisting virus invasion. (3) isolation and identification of PEDV-WN strain. Build animal model. PEDVV was isolated from clinical materials. The M gene of PEDV-WN strain was highly conserved by RT-PCR identification and sequence analysis of MNs gene of PEDV-WN strain, and compared with CV777 strain, the M gene of PEDV-WN strain was highly conserved. A total of 9 amino acid loci were mutated at the core and epitope (CEO 499-638aa), which can be used as the target gene for PEDV detection. The typical animal model of PEDV infection was successfully constructed by regression of PEDV-WN strain. (4) Lactobacillus saliva (Lactobacillus saliarius) inhibited PEDV-WN strain and protected piglets infected by the virus. The preventive and therapeutic intervention of the strain can improve the survival rate of piglets, improve the growth performance of piglets, alleviate the pathological damage caused by the virus to the intestinal tissue, and reduce the proliferation of the virus in the body. Probiotics can regulate the expression of IL-4, IL-8, IL-12, INF- 伪 and MHC 鈪，
本文编号：2172933
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