小肠上皮细胞OATP1A2和OATP2B1载体介导的蛋清源活性寡肽完整吸收机制

The transport mechanisms of food-derived bioactive oligopeptides through the intestinal epithelium in intact forms becomes to be a critical issue that influencing the development of the industry of nutritional food based on food-derived bioactive peptides. Our previous study reveals that organic anion transporting polypeptides OATP1A2 and OATP2B1 may take responsibility for the transport of oligopeptides in intact forms through the intestinal epithelium. The objective of this project is to investigate the OATP1A2 and OATP2B1 mediated transport of egg white-derived bioactive oligopeptides in the model with overexpressed of OATP1A2 and OATP2B1. HPLC/HPLC-MS, RT-PCR and PRM will be used to study the relationship between the intact transport of egg white-derived bioactive oligopeptides and the transcription and expression of OATP1A2 and OATP2B1. Fluorescence labeling and confocal laser microscopy will be used to trace the transport progress of egg white-derived bioactive oligopeptides. It helps to confirm the critical role of OATP1A2 and OATP2B1 in the absorption of oligopeptides in the intestinal epithelium on gene, protein and cell levels. According to the method of transport kinetics, the transport characteristic of oligopeptides, including substrate specificity, H+ or Na+ dependence and cell membrane potential changes will be charactered. Those will help to figure out and explain the transport mechanisms of oligopeptides in the intestinal epithelium mediated by OATP1A2 and OATP2B1. In a word, this work will provide basis for the intact transport of egg white-derived bioactive peptides in the intestinal epithelium mediated.

食源性生物活性寡肽在小肠上皮的完整吸收机制是活性肽研究领域亟待解决的关键问题，制约着活性肽类营养食品的研究和发展。本课题组前期研究表明有机阴离子转运多肽载体OATP1A2和OATP2B1可能参与寡肽在小肠上皮的完整吸收。本项目以蛋清源活性寡肽为对象，构建高表达OATP1A2/OATP2B1细胞模型，利用HPLC-MS、RT-PCR和PRM技术研究蛋清源活性寡肽的完整吸收与OATP1A2/OATP2B1表达之间的关系，利用荧光标记与共聚焦激光显微镜技术研究蛋清源活性寡肽的完整吸收过程，从基因、蛋白和细胞水平确证小肠上皮细胞OATP1A2和OATP2B1对蛋清源活性寡肽的完整转运作用；利用动力学方法研究寡肽完整吸收的结构选择性、离子依赖性和细胞膜电位变化等特性，阐明小肠上皮细胞OATP1A2和OATP2B1介导的蛋清源活性寡肽完整吸收机制。本项目将为蛋清源活性寡肽在小肠上皮的完整吸收提供依据。

本项目旨在探究蛋清肽的肠道吸收机制。首先，利用模拟胃肠道消化法制备蛋清肽，采用Caco-2细胞单层膜和外翻大鼠肠囊模型以及大鼠体内实验系统评价了蛋清肽的肠道吸收，鉴定了大量可完整吸收的蛋清肽结构。然后，以Caco-2细胞模型，分别测定在完全培养基和氨基酸缺乏培养基条件下蛋清肽对有机阴离子转运载体OATP1A2和OATP2B1编码基因SLCO1A2和SLCO2B1转录的影响，结果显示，1~10 mg/mL的蛋清肽对SLCO1A2和SLCO2B1的mRNA水平均无显著影响。但是，相比于完全培养基组，氨基酸缺乏组SLCO1A2和SLCO2B1的mRNA水平均显著上升，说明OATP1A2和OATP2B1可能倾向于在外界氨基酸缺乏时发挥主动转运氨基酸和肽作用，而在外界氨基酸充足条件下，其对蛋清肽的转运作用不显著。在此基础上，进行RNA-sequencing分析，结果发现，在完全培养基条件下，与对照组相比，蛋清肽干预组中有637个上调基因，577个下调基因，KEGG信号通路富集分析结果显示，信号通路endocytosis [KEGG: 04144]最显著，包含12个差异表达基因。其中，基因SMURF1、HSPA1A、HSPA1B、HSPA1L、HSPA2、HSPA6、DNAJC6、RAB31和KIF5A分别编码E3 ligase、Hsc70、auxilin、Rab22和KIF5均显著上调，且参与clathrin蛋白依赖的胞吞途径，包括促进clathrin包被的囊泡形成、囊泡去包被、囊泡回收等过程，该结果与RT-PCR实验结果一致。但是，蛋清肽对clathrin蛋白的转录和表达无显著影响。另外，研究发现蛋清肽能够显著促进肠炎大鼠肠道紧密连接蛋白表达并改善其营养状态。综上所述，通过本项目研究，揭示了蛋清混合肽以clathrin蛋白依赖胞吞为主的肠道吸收机制，为蛋清肽的研究和应用奠定了基础。