【作者】 刘倩；

【导师】 曾东； 倪学勤；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2018， 硕士

【摘要】 胃肠道疾病严重影响了圈养和野生大熊猫的身体健康,研究和开发大熊猫源功能性益生菌对保护大熊猫健康有着重要的理论意义和应用价值。我们已经从健康大熊猫的粪便中成功分离得到了一株益生乳酸杆菌,它具有良好的耐酸、耐胆盐和抑菌作用,并能够有效改善小鼠肠道微生态、提高肠道屏障功能及免疫调节作用。本试验利用粪便移植技术建立大熊猫源化小鼠模型,探究大熊猫源功能性益生菌在模型中的益生作用。1)利用抗生素建立伪无菌小鼠模型,利用粪便移植技术建立菌群大熊猫源化小鼠,采用PCR-DGGE和qPCR技术分析大熊猫源化小鼠的粪便菌群结构和其与大熊猫粪便菌群结构的相似性,以及菌群大熊猫源化后肠道菌群的变化情况,以此来评估大熊猫源化小鼠模型的建立情况。对小鼠连续灌喂抗生素3天后(万古霉素、新霉素和奥美拉唑),灌喂大熊猫粪便菌悬液3天,建立大熊猫源化小鼠模型。检测大熊猫粪便和粪便移植后小鼠菌群变化情况。结果表明,在短期内大熊猫源化小鼠的粪样中肠球菌属的数量极显著降低,厚壁菌门显著升高,梭菌属XIVa、乳杆菌属和瘤胃球菌属极显著升高,而总菌、拟杆菌门、肠杆菌科和双歧杆菌属的数量无显著性差异;大熊猫源化小鼠的粪样菌群和大熊猫粪样菌群的图谱聚类分析相似性约为0.47。这些结果表明,大熊猫源化小鼠模型可在一定程度上代表大熊猫粪样菌群结构,可用于模拟大熊猫肠道内环境进行在体试验。2)利用产肠毒素大肠杆菌作为病原菌侵袭机体,研究大熊猫源功能性益生菌——植物乳酸杆菌BSGP201683在大熊猫源化小鼠模型体内的益生作用。在大熊猫源化小鼠模型的基础上,将小鼠分为4组,空白组(PFMT组)、乳酸杆菌对照组(PFMT-G组)、乳酸杆菌试验组(PFMT-GE组)和大肠杆菌对照组(PFMT-E组)。试验期间,PFMT组连续灌喂PBS溶液18天;PFMT-G组连续灌喂L.plantarum G83 15天后再次灌喂PBS溶液3天;PFMT-GE组续灌喂L.plantarum G83 15天后再次灌喂3天产肠毒素大肠杆菌(ETEC);PFMT-E组连续灌喂PBS溶液15天后再次灌喂3天ETEC。检测回肠组织病理、回肠细胞因子和紧密连接蛋白m RNA表达水平以及粪样菌群变化情况。结果显示,感染大肠杆菌后,与PFMT组相比,PFMT-G组、PFMT-GE组和PFMT-E组的总菌和拟杆菌门含量显著升高;PFMT-GE组的厚壁菌门含量显著高于PFMT-G组;PFMT-GE组和PFMT-E组的梭菌属XIVa含量显著高于PFMT-G组;PFMT-GE组和PFMT-E组的肠杆菌科的含量显著高于PFMT组和PFMT-G组;各组间肠球菌属的数量无显著性差异;PFMT-G组的乳杆菌属含量极显著高于PFMT组、PFMT-GE组和PFMT-E组;PFMT-G组和PFMT-GE组的双歧杆菌属含量极显著高于PFMT组;PFMT组、PFMT-GE组和PFMT-E组的瘤胃球菌属的含量极显著高于PFMT-G组。灌喂乳酸杆菌可以显著提高乳杆菌属、双歧杆菌属和拟杆菌门的含量,并显著降低梭菌属XIVa、肠杆菌科和瘤胃球菌属的含量。提高肠道紧密连接蛋白和细胞因子的分泌,参与并促进炎症发育。这些结果表明植物乳酸杆菌可以改善大熊猫源化小鼠模型的肠道微生态,并对其肠道免疫具有调节作用。更多还原

【Abstract】 Gastrointestinal diseases seriously affect the health of captive and wild giant pandas(Ailuropoda melanoleuca).The research and development of the functional probiotics of the giant panda has important significance and value,in theoretical research and practical application,in protecting the health of the giant panda.We have obtained a potential probiotic strain,Lactobacillus plantarum BSGP201683(L.plantarum G83),isolated from the feces of health giant panda.The microbe survived well at low p H,was tolerant to high bile-salt concentrations,and resistant to antibiotics and pathogenic bacteria.And it has been proved that the microbe also could effectively improve the intestinal microflora,intestinal barrier and attenuate inflammation of mice.This study aimed to evaluate the probiotic effect of L.plantarum G83 in the model of the feces of giant panda transplating to the gut of mice,using the feces of health giant panda,feces microbiota transplation and pseudo mice.To evaluate the model,we analyzed the fecal microflora structure similarity between mice of the model and giant panda by using the PCR-DGGE and qPCR.Mice were treatmented with antibiotic(Vancomycin,Neomycin and Omeprazole)3 d,and orally administrate the bacteria suspension of panda feces 3 d.The results showed that,in the feces of the model mice,the number of Enterococci decreased significantly,Firmicutes,Fusobacterium XIVa,Lactobacillus group and Ruminococcus increased significantly,total bacteria,Bacteriidetes,Enterobacteriaceae family and Bifidobacterium spp.had no significantly change.Cluster analysis of feces flora in panda and model mice was 0.47.These results demonstrated the gut microflora of the model mice could represent panda gut in some extent,and indicated the experimental results in the giant panda.To evaluate the probiotic effects of L.plantarum G83,isolated from giant panda feces,on the model mice when ETEC invasion.Each group was given different treatments as follows:(1)oral administration of sterile PBS 18 d(PFMT);(2)oral administration of L.plantarum G83 15 d and sterile PBS 18 d(PFMT-G);(3)oral administration of L.plantarum G83 15 d and challenge with ETEC 3 d(PFMT-GE);(4)oral administration of sterile PBS 15 d and challenge with ETEC 3 d(PFMT-E).Ileum histopathology,the expression of cytokine and tight-junction protein,and the change of fecal flora were detected.The results demonstrated that after ETEC invasion,compared with PFMT,the number of total bacteria and Bacteroidetes was significantly increased in PFMT-G,PFMT-GE and PFMT-E;the number of Firmicutes in PFMT-GE was significantly higher than PFMT-G;the number of Fusobacterium XIVa in PFMT-GE and PFMT-E was significantly higher than PFMT-G;the level of Enterobacteriaceae family in PFMT-GE and PFMT-E was significantly higher than PFMT and PFMT-G;the level of Enterococci in each groups was no significant difference;the content of Lactobacillus group in PFMT-G was significantly higher than PFMT,PFMT-GE,PFMT-E;the content of Bifidobacterium spp.in PFMT-G and PFMT-GE was significantly higher than PFMT;the level of Ruminococcus in PFMT,PFMT-GE,and PFMT-E was significantly higher than PFMT-G.Administrating with L.plantarum G83 could improved sigmificantly the number of Lactobacillus group,Bifidobacterium spp.,Bacteroidetes,and decreased sigmificantly the number of Fusobacterium XIVa,Enterobacteriaceae family,Ruminococcus.It also could increase the secretion of tight junction protein and cytokines in the intestinal tract and participate in and promote the development of inflammation.These results suggest that L.plantarum G83 could improve the intestinal microecology of the model mice,and have immune regulation effect on its intestinal immunity.更多还原