基于量子点标记技术的canolol细胞抗氧化活性及作用机理研究

Dietary polyphenols play an important role in human nutrition and health, because they could reduce oxidative damage. Canolol is a new kind of polyphenols in rapeseed and rapeseed oil. Although its antioxidant activity has been proved by some chemical methods, the systematic theories still lack to reflect its real antioxidant activity and the functional mechanisms in organisms, limiting the development of nutritional value. In this project, a platform for the evaluation of cellular antioxidant activity is established to explicit the antioxidant ability of canolol by selecting human hepatoma cell line as a model. And a cell model with oxidative damage is built to explore the evolution of physiological and biochemical indexes, elucidating the relationship of time-concentration-effect, as well as the pathway. Because canolol has no intuitive and visual optical properties, and it is difficult to directly obtain and deduce the movement and distribution in organisms, therefore, canolol-labeled quantum dots would be constructed in this study as a fluorescent probe based on the advantages of quantum dots such as good stability, high quantum yield and strong ability to resist photobleaching, and so on. The movement and distribution of canolol in the cell are tracked and visualized by confocal microscopy imaging, flow cytometry and co-locating technologies. On the basis of the above results, the cellular antioxidant activity and functional mechanisms of canolol are revealed, providing a theoretical support for the development and utilization of canolol and the production of high-value rapeseed.

膳食多酚能降低机体氧化损伤，对人体营养健康发挥重要作用。Canolol是油菜籽和菜籽油中的一种新型多酚，化学法已证实其抗氧化活性，但缺乏其在有机体内的真实抗氧化活性和作用机理的理论支撑，限制了营养学价值的开发。本项目拟以人肝癌细胞株为模型，建立细胞抗氧化评价平台，明晰canolol的细胞抗氧化能力；构建氧化损伤细胞模型，探究细胞生理生化指标的演变规律，阐明抗氧化的时量效关系及作用途径；由于canolol不具备直观可视化的光学性质，针对其在有机体内的作用过程和分布难以直接获知或推导的现状，本研究拟利用量子点稳定性好、量子产率高、抗光漂白能力强等优点，构建量子点标记的canolol荧光探针，运用共聚焦显微成像、流式细胞和共定位等技术，示踪canolol摄入细胞的过程，可视化其在细胞内的作用分布。基于以上结果，揭示canolol的细胞抗氧化活性与作用机理，为其开发与利用及菜籽高值化提供理论依据。

膳食多酚能降低机体氧化损伤，对人体营养健康发挥重要作用。为探明菜籽油多酚canolol在有机体内的真实抗氧化活性和作用机理，本项目以HepG2细胞为研究模型，采用细胞抗氧化CAA法、细胞毒性MTT和亚甲基蓝法评价了canolol细胞内抗氧化活性和细胞毒性。研究表明，无PBS清洗条件下，canolol抗氧化能力高于白藜芦醇和芝麻酚，略低低于芥子酸；PBS清洗条件下，canolol细胞抗氧化能力强于芝麻酚，略低于白藜芦醇和芥子酸，但是canolol对细胞的毒性远低于上述三种油料多酚。H2O2诱导的HepG2细胞氧化应激损伤模型研究结果表明，1-100 μM canolol 预处理细胞后明显抑制H2O2诱导的细胞存活率降低，ROS含量、LDH和MDA水平增加，恢复细胞形态，显著提高CAT、SOD和GSH水平。JC-1染色、流式细胞术、蛋白印迹实验结果显示，canolol阻止细胞线粒体膜电位下降，膜电位值从45.5%提升至77.5%，抑制H2O2诱导的细胞凋亡，逆转H2O2诱导的Bax/Bcl-2比值和caspase-3蛋白表达增加，减缓H2O2诱导的p38蛋白磷酸化。Canolol通过p38 MAPK信号通路抑制氧化应激诱导的细胞损伤。为明晰canolol在细胞内的分布，利用有机合成、化学偶联等技术制备红色量子点标记的canolol荧光探针。研究发现，canolol荧光探针细胞毒性较低，且对canolol细胞抗氧化活性影响较小。荧光共定位结果显示，canolol分布在细胞核周围。研究探明了canolol细胞抗氧化活性，阐述了canolol的抗氧化量效关系，分子机制和作用分布。项目按计划圆满完成了研究任务，相关结果在高水平期刊上发表SCI文章8篇，超额完成原定1-2篇的任务，申请国家发明专利1项。研究结果为canolol开发与利用及菜籽高值化提供理论支撑。