仔猪肠粘膜上皮细胞与细菌的铁竞争机制及其在免疫调节中的作用研究

In this study, real time PCR and western-blotting will be used to screen the factors in iron competition between host cells and bacteria. The RNA interference and neutralization assay will be performed to analyze the key factors of host cells involved in the iron competition. The screened iron competition related genes of bacteria will be knockd out by homologous recombination technique.The deficient mutant will be used to determine the bacterial iron competition factors by analazing the abilities of iron uptake of bacteria. On the basis of revealing the iron competition mechanisms, the real time PCR, western-blotting, ELISA, RNAi and gene knockout by homologous recombination techniques will be used to study the roles of iron competition factors in the immunological regulation of host cells,adherence and colonization of probiotics and the expression of virulence factors of pathogenic bateria. These studies will supply new clues for the mechanism of immunological response stimulated by bacteria. After animal experiments, the PCR-DGGE, real time PCR and ELISA will be applied to study the effects of dietary iron on the piglet gut bacterial composition, the expressional levels of host iron competition proteins and immunological functions. The roles of dietary iron during the interaction between intestinal epithelial cells and micro-organisms will be revealed. This study will provide new ideas for the development of probiotics and prevention and treatment of pathogenic bacteria.

首先运用荧光定量PCR、Western blotting等技术，初筛宿主细胞及细菌参与铁竞争的蛋白因子。再以RNA干扰、抗体中和分析等技术，解析宿主细胞铁竞争因子的作用机制；并以同源重组基因敲除技术，获得细菌铁代谢基因缺陷突变株，通过分析其铁摄取能力的变化，解析细菌铁竞争因子的作用机制，从而阐明宿主细胞与细菌的铁竞争机制。在阐明宿主细胞与细菌铁竞争机制的基础上，综合运用荧光定量PCR、ELISA、RNAi、同源重组基因敲除等技术，探究铁竞争机制在宿主细胞免疫调节、益生菌粘附定植及致病菌毒力因子表达中的作用，为揭示细菌刺激肠粘膜细胞免疫反应的机制提供新线索。通过动物实验，运用PCR-DGGE、荧光定量PCR及ELISA等技术，分析日粮铁水平对仔猪肠道菌群组成、机体铁竞争因子表达水平及免疫机能的影响，探究日粮铁在宿主细胞与细菌互作中的作用，为新型益生菌的开发及致病菌的防治提供新思路。

本研究利用生长曲线法，证明了铁能够刺激致病性大肠杆菌（enterohemorrhagic E.coli , EHEC）的生长、增殖，对植物乳杆菌（Lactobacillus plantarum）这一益生菌则没有明显影响。利用双向电泳和液-质联用进行的蛋白质组学研究证明，铁能够明显刺激EHEC蛋白的表达谱明显改变，而对益生菌的蛋白质表达谱影响较小。利用RT-PCR进行的研究证明，培养基中的铁能够刺激致病性大肠杆菌Fur因子及FhuA、FepA、FecA、TonB等铁摄取相关蛋白质的表达上调，这提示该菌主要通过Fur调节的摄取机制进行铁摄取，其中FepA、FecA、TonB等转运蛋白及TonB-ExbB-ExbD系统组成的嗜铁素摄取系统是其重要的铁摄取方式。RT-PCR和Western Blotting结果表明，宿主细胞通过转铁蛋白受体（TFR）和LCN-2的作用摄取培养基中的铁。本研究利用RT-PCR和ELISA技术，证明了铁能够促进eaeA和LER等致病性大肠杆菌毒力因子的表达上调，增强其毒力，进而激活宿主细胞NF-κB信号通路，诱发炎症。动物试验则表明，日粮添加80-160mg/kg铁时，随着铁水平升高，仔猪盲肠中大肠杆菌、沙门氏菌等致病菌的数量增加，而双歧杆菌、乳酸杆菌等益生菌的数量下降；同时，高浓度的日粮铁还会引起仔猪空肠粘膜IL-1β、IL-6、IL-12和TNF-α等炎症因子的水平上升，导致仔猪肠道发生炎症。