基于适配体荧光纳米探针的呕吐毒素细胞毒性成像分析研究

真菌毒素严重污染粮食食品，极大地危害人体健康。探索真菌毒素毒性效应和作用机制是食品安全重要的研究方向。为解决食品毒理学和细胞分子生物学在真菌毒素毒理研究中的共性问题，本项目立足于多学科交叉融通，以呕吐毒素（DON）为研究对象，通过SELEX技术筛选DON及毒性通路中关键靶点（caspase-3）的适配体序列，合成光谱可辨的上转换纳米粒子，构筑适配体荧光探针。研究适配体探针跨膜递运机制，通过化学修饰提高其胞内稳定性。以人肝癌细胞（HepG2）为模型细胞，通过适配体原位捕获和多色上转换荧光标记，对胞内靶标进行实时动态成像监测。依据成像信息解析毒素“剂量-时空”分布特征，揭示毒性通路各靶点之间应答关系，阐明毒性效应及分子作用机制，进而建立细胞毒性可视化成像评价体系。本项目提出基于适配体荧光纳米探针的细胞成像分析可为真菌毒素的毒理研究提供新模式，有望推动多学科协同发展，对保障人体健康具有积极意义。

Mycotoxins contaminate broad range of grain foods and lead to serious human health problems. Exploring mycotoxins toxicity effect and mechanism is important for food safety. In order to solve the common problems of food toxicology and cellular molecular biology for mycotoxins toxicological research, in this work, based on the interdisciplinarity and accommodation, deoxynivalenol (DON) is chosen as the typical mycotoxin. Aptamers against deoxynivalenol and the key target (caspase-3) in toxicity pathway would be selected through Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Upconversion nanoparticles (UCNPs) with multispectral emissions would be synthesized and functionalized with aptamers to form fluorescence probes. The transmembrane mechanism of the probes would be investigated. The resistance to enzymatic degradation of the probes would be improved by chemical modifications. Human liver cancer cell (HepG2) is used as the target cell, real-time and dynamic monitoring of targets in HepG2 through aptamer in situ capturing and multi-color UCNPs labeling would be achieved. The distribution characteristics of “dose-space time” of DON would be analyzed according to the imaging information. The response relationship among each target in toxicity pathway would be revealed. The toxicity effect and mechanism would be clarified, and the visualization evaluation system for cellular cytotoxicity would be developed. The cell imaging platform based on aptamer functionalized fluorescence probes can provide a new mode for toxicological research of mycotoxins, and is significant to facilitate the coordinated development of multiple disciplines and human health.