基于Caco-2细胞膜和大鼠外翻肠囊模型的蛋清源活性寡肽完整吸收途径及机制研究

To exert bioactivity in vivo, egg white-derived bioactive peptides, i.e. ACE-inhibitory peptides, must be absorbed in intact form. It is known that the most of bioactive peptides identified from food protein have more than three amino acids, but their absorption mechanism is still obscure. This project aims to investigate the transport of egg white-derived oligopeptides through intestinal epithelium using Caco-2 cell monolayers and everted rat intestinal sacs. First, the peptides that can be absorbed in intact form will be identified. Based on the absorbed peptides, as well as artificial designed oligopeptides, the relationship between the structure and transport of peptides will be studied. Second, the physiological properties, including transport kinetic characteristics, dependence to pH gradient and sodium salt, dynamic changes of intracellular pH value, potential and membrane potential and so on during the transport process of transport of oligopeptides will be determined. Third, confocal laser scanning microscopy and transmission electron microscopy are used to observe the transport route and process of some oligoppetides that labled with fluorescent molecules. Some transport inhibitors will be also used to verify the transport pathways of oligopeptides. Moreover, the models of enterocytes or rats that incubated with high concentration of egg white-derived oligopeptides will be established. Then, the differentially expressed genes and proteins compared to control group will be identified using gene chips and 2-D gel electrophoresis. It is possible to provide evidences and explore the biological mechanism of the transport of egg white-derived oligopeptides from celland molecular levels. This project is expected to contribute the understanding of the intact transport of food derived bioactive oligopeptides.

蛋清源活性肽如ACE抑制肽等要在体内发挥活性，须以其完整形式被小肠吸收进入体内。目前鉴定的活性肽长度一般在3个氨基酸以上，然而这部分寡肽的体内完整吸收性及吸收机制尚不明确，是活性肽研究领域急需解决的关键问题。本项目利用Caco-2细胞膜和大鼠外翻肠囊模型，鉴定蛋清源可完整吸收的寡肽，结合人工设计肽序列，明确肽的结构与完整吸收关系；研究蛋清寡肽完整吸收的动力学特征，及其对pH梯度、Na+浓度的依赖性，以及完整吸收过程伴随的细胞内pH、电位和细胞膜电位的动态变化等生理特性；利用荧光标记技术，通过共聚焦激光显微镜和透射电子显微镜观察寡肽的完整吸收途径和过程，结合吸收通路抑制剂，明确寡肽的完整吸收通路，并以高浓度蛋清寡肽胁迫处理肠细胞或灌胃大鼠，分析差异表达基因和蛋白，从细胞和分子水平阐述蛋清寡肽的完整吸收途径和机制。本项目研究可为寡肽的小肠完整吸收提供证明，为活性肽的科学研究和加工应用奠定基础。

蛋清源活性肽要在体内发挥活性，须以完整形式被小肠上皮吸收进入血液循环，但长度大于3个氨基酸的寡肽，其完整吸收特性及机制尚不明确，是活性肽研究领域急需解决的科学问题。本项目利用Caco-2细胞膜和大鼠外翻肠囊模型，对226个分子量小于1 kDa的蛋清肽进行了完整吸收实验及吸收效率评价；结合人工设计肽序列，明确了蛋清肽完整吸收的构效关系；研究了蛋清寡肽完整吸收的理化特性（亲疏水性和分子量等），探讨了酶解方式、温度对蛋清源寡肽完整吸收的影响；研究了蛋清肽对调控紧密连接蛋白表达的影响，解析了蛋清源寡肽完整吸收的分子机制；最后研究了蛋清肽诱导脂溶性组分在多糖体系下的共组装机制及吸收增益特性。本研究可为食源性活性寡肽的小肠完整吸收提供依据，为活性肽的科学研究和加工应用奠定理论基础。.主要结果：1.分别有56条和28条分子量低于1 kDa的蛋清肽能够被Caco-2 细胞单层膜和外翻大鼠肠囊完整吸收（主要来源于卵白蛋白、卵转铁蛋白和溶菌酶）。其中，4条蛋清肽能同时被两种模型完整吸收（LGAKDSTRT、VNDLQGKTS、DGSRQPVDN、GKKDPVLKD）。2.N末端氨基酸残基对寡肽Caco-2细胞膜吸收的影响大于C末端，N末端替换后的多肽吸收率相对较高。寡肽可能存在两类跨膜吸收途径，一是受紧密连接调控的细胞旁路途径，易于转运亲水性、分子量小的寡肽，二是寡肽载体转运和胞吞两种跨细胞途径，易于转运输水性强的寡肽。3.温度不会影响细胞旁路途径但会抑制转运载体和胞吞途径，与体外模拟胃肠消化相比，碱性蛋白酶酶解可促进蛋清源寡肽的完整吸收，但不利于旁路途径吸收。4.碱性蛋白酶酶解产物通过促进紧密连接相关蛋白的表达，锁紧旁路渗透途径通道，增大细胞跨膜电阻，因而不利于蛋清肽通过旁路途径吸收。另一方面，碱性蛋白酶酶解产物可能对小肠损伤起到较好的修复作用。5.蛋清肽能够协同多糖类化合物提升疏水性活性物质的水溶性和活性。同时，部分蛋清肽能够通过被动扩散和旁路渗透等方式被完整吸收。