肠细胞ASBT介导的脱氧胆酸偶联壳聚糖-蛋清源活性肽纳米颗粒胞吞作用机制

蛋清源活性肽因其良好的生物活性被广泛关注，但由于小肠完整吸收率低而限制了应用。研究显示脱氧胆酸偶联壳聚糖纳米颗粒可显著提高活性肽的小肠吸收，但其作用机制尚不明确。本项目以蛋清源活性肽为对象，构建脱氧胆酸偶联壳聚糖-蛋清肽纳米颗粒，阐明该纳米颗粒的结构和对ASBT（顶端钠依赖性胆酸转运体）亲和性与蛋清肽完整吸收之间的关系；利用蛋白质组学和免疫荧光研究脱氧胆酸偶联壳聚糖-蛋清肽纳米颗粒对ASBT结构、功能及位置迁移的调控作用；采用胶体金标记揭示脱氧胆酸偶联壳聚糖-蛋清肽纳米颗粒胞吞作用过程；运用免疫共沉淀和免疫荧光明确ASBT介导的脱氧胆酸偶联壳聚糖-蛋清肽纳米颗粒胞吞过程中的囊泡形成机制，从分子和细胞水平上阐明ASBT介导的脱氧胆酸偶联壳聚糖-蛋清源活性肽纳米颗粒胞吞作用机制。本项目将为基于脱氧胆酸偶联壳聚糖纳米颗粒的促进蛋清源活性肽小肠吸收策略奠定基础，为蛋清源活性肽的研究和应用提供支撑。

Egg white bioactive peptides have attracted broad attention for their bioactivity, but the application is limited due to the low intestinal permeability. Studies show that deoxycholic acid conjugated chitosan nanoparticles can significantly increase the permeability of bioactive peptides in the enterocyte, but its transport mechanism is still unclear. Therefore, this research aims to fabricate deoxycholic acid conjugated chitosan-egg white bioactive peptide nanoparticles, and characterize the relationship among nanoparticles structure, affinity to ASBT (apical sodium-dependent bile acid transporter) and the intact absorption of egg white bioactive peptides. Furthermore, the regulation of deoxycholic acid conjugated chitosan-egg white bioactive peptide nanoparticles on the structure, function, as well as position migration of ASBT is clarified by using proteomics and immunofluorescence. Meanwhile, the colloidal gold labels help to reveal the endocytosis process of deoxycholic acid conjugated chitosan-egg white bioactive peptide nanoparticles, and co-immunoprecipitation and immunofluorescence are used to clarify the mechanism of vesicle formation induced by ASBT-mediated deoxycholic acid conjugated chitosan-egg white bioactive peptide nanoparticles. This research elucidates the mechanism of ASBT-mediated deoxycholic acid conjugated chitosan-egg white bioactive peptide nanoparticles endocytosis at the cellular level. This study tries to lay a foundation for the intestinal epithelium absorption strategy of egg white bioactive peptides based on deoxycholic acid conjugated chitosan nanoparticles, and provide support for the research and application of egg white bioactive peptides.