副干酪乳杆菌S-层蛋白黏附特性及对肠道屏障功能保护作用研究

Enteropathogens infection is a cause of intestinal disease in humans by disrupted the Intestinal barrier function. Antibiotics treatment appears to increase the risk for pathogens-drugtolerance. Application of probiotics lactobacilli to prevent pathogen from adhesion and modulate intestinal barrier function has received much attention due to safety and the S-layer proteins play the importane role. But the underlying mechanisms remain unclear for the S-layer proteins of lactobacilli prevention the epithelial barrier dysfunction and modulation the NF-κB and MAPK singaling pathways, this limited the utilizition and research on the S-layer proteins of lactobacilli. Our research group have selected the strain of L. paracasei M5-L, which can inhibite the adhesion of S.sonnei to HT-29 cells and the S-layer proteins playing the importane role in inhibition of S.sonnei adhesion. Base on this, we will employed the methods of MALDI-TOF to investigate the properties of surface layer proteins of L. paracasei M5-L, and associated with adhesion of surface layer proteins to HT-29 is dectected in the SDS-PAGE and Western blotting with NHS-Biotin labeled HT-29 cells, Atomic Force Microscope analysis and adhesion structure region analysis. In addition, the S-layer proteins of L. paracasei M5-L prevente the epithelial barrier dysfunction and regulate the NF-κB and MAPK singaling pathways are detected with the method of RT-PCR,Western blotting, immunofluoresence and ELISA， this research will be an important value.

致病菌感染后通过破坏肠上皮细胞的屏障功能引发炎症性肠病，耐药菌株的出现给抗生素治疗带来了风险。乳酸杆菌在人体肠道内抑制致病菌黏附、改善肠道屏障功能具有安全性，其中的S-层蛋白发挥了重要作用。但目前有关乳酸杆菌S-层蛋白对肠道屏障功能的调节作用和机制缺乏系统的研究，限制了S-层蛋白的开发和利用。课题组前期筛选到L. paracasei M5-L能够有效抑制S.sonnei对HT-29细胞的黏附，其中的S-层蛋白发挥重要作用。在此基础上，拟采用MALDI-TOF研究其 S-层蛋白性质；NHS-Biotin标记细胞与SDS-PAGE 中的S-层蛋白进行杂交、原子力显微镜和黏附附结构域分析确定S-层蛋白黏附作用机制；采用RT-PCR、Western Blott、荧光免疫组化和ELISA等手段研究S-层蛋白对上皮细胞屏障功能防护作用及其信号途径的调节作用，阐明其作用机制，具有重要价值。

本课题通过LC/Q-TOF MS/MS质谱分析技术鉴定得到M5-L分离出来的蛋白主要为表面抗原，在此基础上通过NHS-Biotin标记细胞与SDS-PAGE 中的S-层蛋白进行杂交、原子力显微镜分析证明S-层蛋白具有黏附作用；采用RT-PCR、Western Blott、荧光免疫组化和ELISA等手段研究S-层蛋白对上皮细胞屏障功能防护作用及其信号途径的调节作用。100 μg/mL的 S-层蛋白能够抑制致病菌对上皮细胞的黏附及其增殖作用，同时S-层蛋白能够抑制致病菌S. sonnei引起Occludin和ZO-1蛋白减少的作用，即M5-LS-层蛋白在对细胞的保护中起预防作用。通过ELISA测定细胞分泌的IL-8、IL-10、TNF-α、IL-1β的水平及RT-PCR对相应细胞因子的mRNA表达情况的研究，证实了两种S-层蛋白能够在基因及蛋白水平诱导细胞产生抗炎性细胞因子IL-10，同时能够在基因及蛋白水平拮抗S. sonnei刺激细胞分泌的促炎性细胞因子IL-8、TNF-α、IL-1β。进一步通过Western Blot法对HT-29细胞的MAPK信号通路进行研究，发现S-层蛋白能够通过降低p-JNK蛋白的表达水平，进而拮抗S. sonnei对JNK信号通路的活化作用。项目资助发表SCI论文1篇；EI论文1篇，核心论文4 篇，正在整理英文文章2篇。培养硕士生5名，其中4名已经取得硕士学位，1名在读。项目投入经费23万元，支出19.3569 万元，预算中的测试费大部分调整为材料费。剩余经费3.6431 万元，剩余经费计划用于本项目研究后续支出。