植物乳杆菌减缓仔猪大肠杆菌性腹泻机制研究

口服益生菌是防治肠毒性大肠杆菌引起的儿童和新生动物腹泻的有效手段，其防治机制可能涉及肠道细胞信号通路和屏障功能的调节。本项目首先通过细胞试验，采用免疫组化、western blot等技术研究植物乳杆菌调节对ETEC感染细胞时紧密连接蛋白的表达和分布变化，采用PCR-array研究植物乳杆菌对ETEC感染细胞时NF-κB和MAPK信号通路中各蛋白转录情况的变化的影响，通过western blot分析信号通路中蛋白的表达量或磷酸化变化趋势，通过荧光定量PCR等技术研究这些变化如何引起细胞因子的表达变化进而缓解细胞的炎性反应；随后通过原位小肠结节灌注的及ETEC感染动物模型验证细胞模型上结果，并通过宏基因组学和代谢组学分析肠道微生物区系变化和机体代谢特征。本研究可为探索研究乳杆菌与机体的互作机制提供新的思路，同时为应用益生菌防治动物及新生儿腹泻提供理论依据。

Oral administration of probiotics is an effective method of preventing diarrhea caused by the enterotoxigenic Escherichia coli (ETEC) in children and newborn animals. The underlying mechanism may involve modulations in cell signaling pathways and adjustments in gut barrier function. In a cell model, immunohistochemistry and western blot will be carried out to investigate whether Lactobacillus plantarum adjust and change expression and distribution of tight junction in ETEC infection cells. PCR-array data will also be analyzed to determine transcriptional differences of the genes in NF-κB and MAPK signal pathway in ETEC infected cells. Western blot will also be carried out to detect the trends in expression and phosphorylation of proteins in the signal pathway, and quantitative PCR will be used to determine the changes in cytokine expression and inflammatory response. A following in situ small intestinal segment perfusion model and an ETEC-chanllenged model will also be carried out to determine whether the protein expression in the signal pathway agree with the results of the cell model, further macro genomics and metabolomics analysis will be performed to analyse the intestinal microflora changes and body metabolic characteristics. This research will provide new ideas for the study of mechanism underlying the interaction of Lactobacillus and the host, and provide a theoretical basis for the application of probiotics in prevention and treatment of animal and neonatal diarrhea.