基于荧光探针的紫檀芪食品胶体转运机制研究

Pterostilbene is one nutraceutical with many health promoting activities. But low water solubility has limited its application in food products. The use of colloidal system is one effective approach to solve this problem. However, its transport mechanism across small intestine epithelium cell is not clear, primarily due to lack of intracellular trafficking method. To overcome this, we developed a fluorescent probe for pterostilbene. The transport mechanism of pterostilbene colloidal system across small intestine will be elucidated by using this pterostilbene probe. In this study, pterostilbene will be encapsulated in zein nanoparticle colloidal system. In vitro digestion model will be applied to study the digestion process of this delivery system, while CLSM will be used to monitor the change of pterostilbene during digestion process. After digestion, Caco-2 monolayer will be used to simulate small intestine epithelium cell. The distribution of pterostilbene nanoparticle inside the cell will be monitored by CLSM. The endocytosis and exocytosis pathway of pterostilbene will be identified by applying transport inhibitors. The cellular level of pterostilbene will be determined. The transport mechanism of pterostilbene across the small intestine epithelium will be elucidated by integrating the above-mentioned results. The result of this study will contribute to the rational design of pterostilbene delivery system; the fluorescent probe developed herein will become a powerful tool for study other biological activities of pterostilbene.

紫檀芪是一种具有诸多健康功效的食品功能因子，但它的水溶性较差，不能直接添加到食品中。采用食品胶体进行包埋是解决这一问题的有效手段。但是紫檀芪胶体体系在小肠上皮细胞的转运机制仍不明，特别是针对性的追踪方法有限，因此我们通过前期研究构建了一种紫檀芪荧光探针，拟借助该手段阐明紫檀芪胶体在小肠内的转运机制。本课题拟制备由玉米蛋白纳米颗粒组成的紫檀芪胶体体系；接着进行模拟体外消化，利用激光共聚焦显微镜(CLSM)观察在此过程中紫檀芪纳米颗粒形态的变化；用Caco-2单细胞层模拟小肠上皮细胞，借助CLSM观察紫檀芪纳米颗粒在细胞中的分布、细胞形态的变化；通过添加相应转运抑制剂揭示细胞对紫檀芪纳米颗粒的转运方式；借助定量分析确定胞内紫檀芪水平；最终阐明小肠上皮细胞对紫檀芪胶体转运体系的转运机制。本课题将为紫檀芪转运体系的合理设计提供理论依据，研制的荧光探针将为紫檀芪其他各种活性研究提供有力手段。

紫檀芪是一种具有诸多健康功效的食品功能因子，但它的水溶性较差，不能直接添加到食品中，采用食品胶体进行包埋是解决这一问题的有效手段。但是紫檀芪胶体体系在小肠上皮细胞的转运机制仍不明，特别是针对性的追踪方法有限，因此本课题成功用丹磺酰氯构建了一种紫檀芪荧光探针，质谱实验表明，紫檀芪的羟基通过取代丹磺酰氯分子中的氯与其相连，通过引入发色基团形成荧光络合物。通过反溶剂法制备由玉米蛋白纳米颗粒组成的紫檀芪胶体体系，纳米颗粒平均粒径为120-150nm，接着用进行模拟胃肠道消化，用激光共聚焦显微镜研究了在此过程中紫檀芪纳米胶体体系形态的变化；研究了紫檀芪及其荧光络合物对Caco-2细胞毒性的影响，当样品浓度大于20μM时，紫檀芪荧光络合物细胞毒性比紫檀芪低；用紫檀芪和紫檀芪荧光络合物处理细胞后，只有紫檀芪荧光络合物组观测了荧光，验证了这种荧光追踪方法的有效性；通过添加相应转运抑制剂揭示细胞对紫檀芪纳米颗粒的转运方式；借助定量分析确定胞内紫檀芪水平；研究结果表明Caco-2细胞对紫檀芪胶体转运体系的为网格蛋白介导的内吞过程。本课题将为紫檀芪转运体系的合理设计提供理论依据，研制的荧光探针将为紫檀芪其他各种活性研究提供有力手段。同时本课题的研究内容将为具有类似化学性质的疏水性营养素转运体系的开发和体内吸收研究提供参考。