基于人类M样细胞模型的牛乳酪蛋白肠粘膜免疫调节作用

Food derived proteins are not only a kind of important nutreint, but also physiological regulators.Physiological active proteins and peptides have received increasing attention as potential medicines and ingredients of health-promoting functional foods. It is difficulty to illustrate how the Physiological active proteins and peptides worked after hydrolyzed at all stages of the gastrointestinal tract digestion by animal experiments. In this project an in vitro multi-step static model is designed to simulate complete digestion of bovine milk casein. The aim was to identify and clarify the distribution of the peptide candidates able to exhibit signi?cant immunopotentiation effects after GI digesting. The experimental set-up involved the enzymatic digestion of casein in simulated gastric, duodenal and jejuneal environments to closely reproduce the in vivo conditions. Bioassay-guided isolation is used to fractionate the digests by multichromatographic resolution. The immunoactive peptides was identified by the combined application of HPLC and mass spectrometry techniques. A new approach are suggested to evaluate the absorption of peptides by co-culture of Caco-2 and B cell line to form an M-cell model in vitro and to demonstrate the absorption mechanisms in cell level. This model are used to study the regulation of mucosal immune system by peptides derived from the digests and illustrate the immune regulation network influenced by the food protein. The digestion method and cell evaluation will be integrated into a new system to study bioactive proteins and peptides mostly close in vivo conditions. The achievements will play a significant role in the net regulation studying nutrition and physiological active effect of proteins and peptides.

食物蛋白不仅具有营养功能，其降解产生多种活性多肽已成为食品科学的研究热点。但这些多肽的生理调节功能往往以动物或人体整体考查，难以深究功能因子经消化道消化、转运吸收及发挥作用的机制。本研究以牛乳酪蛋白为模型蛋白，模拟胃、肠环境消化过程，分级分离并鉴定消化过程中产生的多肽分布、种类与结构，辅以实验动物消化液分析验证，阐明酪蛋白及其活性降解物在胃肠的生理作用物质基础。提出建立人Caco-2及与B淋巴细胞共培养模拟人小肠M样细胞，在细胞水平上揭示活性蛋白及其降解多肽的吸收形式、转运机制，寻找促进吸收方式的新思路，通过M细胞模型中B细胞免疫因子的检测，揭示活性多肽对粘膜免疫的调节机制。最终整合形成活性蛋白因子转运吸收体外评价体系。该研究对促进活性多肽作用机制及评价方法具有重要意义，对食物在体内营养生理调节、免疫信号传递通讯网络具有重要参考价值。

食物蛋白不仅具有营养功能，体外降解可产生多种生理功能活性多肽，如牛乳酪蛋白降解产生ACE抑制活性肽、免疫活性肽、抗疲劳肽等。酪蛋白经胃肠水解是否也能产生相应的生理功能活性肽段，这些肽段是否能被吸收发挥生理功能值得关注。. 本项目对牛乳酪蛋白进行了胃肠模拟水解，并对水解不同时间的肽段活性和分布检测发现，酪蛋白在模拟胃肠联合内水解2h后，ACE抑制率为45.8%，DPPH自由基清除率为46.4%，对大肠杆菌和金黄色葡萄球菌均有抑制作用，对金黄色葡萄球菌的抑制效果更强。建立较稳定的RP-HPLC分析方法，用于胃肠模拟水解液和动物胃肠消化液分析，两种水解液的相似度在80%以上。模拟水解物的质谱（LC-MS/MS）分析显示水解物中确实存在已证实具有ACE抑制活性及免疫活性肽段。.建立了Caco-2细胞单层膜模型，首先用于酪蛋白水解物的跨膜转运研究，发现亲水性多肽相比疏水性多肽更易通过单层细胞膜。由于水解物肽段复杂，因此研究了合成酪蛋白源ACE抑制肽的转运机制。合成肽在5mmol/L对Caco-2细胞的增殖没有影响，在pH7.4时转运量最大，随着转运时间和浓度的增大，转运呈增加趋势。在该条件下，500μmol/L的RYLGY转运90min，BL侧测得的量约为8.3%。采用三种不同长度合成肽混合跨膜转运时，内吞作用不显著，而去氧胆酸钠能显著增加RYLGY及LHLPLP或WQVLPNAVPAK及RPKHPIKHQ的旁路转运量，表明主要这几种酸性肽主要通过细胞间的连接被转运。叠氮化钠和维拉帕米能显著增加RYLGY及LHLPLP或WQVLPNAVPAK的量，在多肽转运吸收过程中由P-糖蛋白介导的外排作用显著。. 对酪蛋白进行FITC荧光标记后进行大鼠体内肠吸收的追踪分析，灌胃3h后发现，能够进入粘膜下层（即通过肠上皮细胞），血管壁显示较显著荧光，FITC-酪蛋白水解物能够被吸收，吸收形式有待确定。本项目明确了酪蛋白体内水解确实能产生生理活性肽类，且主要通过肠上皮细胞连接跨膜转运，能保留原有活性形式，为促进活性多肽作用机制研究及评价方法提供参考，丰富人们对了食物在体内营养生理调节作用的理解。