肿瘤细胞上皮间质转化过程中乏氧与活性氧的近红外荧光检测及其可视化研究

Mitochondrial reactive oxygen species (ROS) produced by hypoxia can regulate epithelial-mesenchymal transitions (EMT) of tumor cells, but the method to probe endogenous ROS (<nM) has not yet been established. Considering that upconverting nanoparticles (UCNPs) have great advantages in photochemical stability, quantum efficiency, signal-to-noise ratio, and that the fluorescent dye embeded in nano materials has better photochemical stability and higher quantum efficiency, a hybrid functionalized nanoprobe will be designed and assembled, which is composed of polypeptide (cell penetrating peptide or targeted mitochondria peptide), polymer as carrier, UCNPs as energy donors and two dye-based probes as energy acceptors. The assembled nanoprobe should be more sensitive and selective than dye-based probes for O2 and/or ROS, and be more suitable for simultaneously detecting two different target molecules. In addition, a near-infrared fluorescent probe for O2 will be also designed and synthesized with lipophilic cation dye having mitochondria- targeted characteristic and nitroimidazole being a potential hypoxia reporter. Based on these probes mentioned above and an anti-E-cadherin/FITC, it will be very possible to perform simultaneously detecting and imaging O2 and/or E-cadherin and/or ROS produced by hypoxia in the epithelial-mesenchymal transitions of tumor cells, and then to achieve some correlation information on ROS-mediated EMT pathway. These results are intended to provide new strategies and new ways to deeply investigate the molecule mechanism of hypoxia-induced EMT and other ROS-mediated signal transduction pathway.

乏氧诱导线粒体活性氧产生，进而参与介导肿瘤细胞上皮间质转化（EMT）过程。本项目基于上转换纳米材料具有光稳定性好、量子效率高、单激发双发射及信号放大、信噪比高等特点，将NaYF4:Yb, Er上转换纳米颗粒与两种有机小分子荧光探针（检测活性氧或者乏氧）通过嵌段共聚物组装，构建一类多肽-聚合物-上转换纳米颗粒-有机小分子探针的复合功能化荧光探针。该类探针会极大提高检测的灵敏度与选择性。另外，拟以具有靶向线粒体功能的亲脂性阳离子染料及对乏氧环境敏感的硝基咪唑类活性基，设计合成线粒体内检测与成像分子氧的近红外荧光探针。实现高选择性、高灵敏度、同时检测乏氧肿瘤细胞内分子氧、信使活性氧的新方法，以及分子氧、活性氧、E-cadherin蛋白（EMT标志性蛋白）的同时成像分析。其结果为乏氧诱导的EMT过程的分子机制研究及其他活性氧介导的信号通路研究提供新的策略与新的途径。

本项目以肿瘤细胞上皮间质转化过程中乏氧与活性氧检测方法的构建为切入点，开展了超高灵敏活性氧及还原性分子荧光可视化分析方法的研究。基于纳米材料与有机小分子的组装、环化扩展Stokes位移、含Se-N键五元环的开关调控及特异性报告基团的发现，我们发展了一系列高灵敏、高选择性的活性分子的荧光探针，分别对特定的分子事件过程中其关键活性分子进行了细胞及活体内的实时成像分析。1）设计合成了一种检测乏氧程度的近红外荧光探针，并用此探针研究了肿瘤细胞乏氧程度与E-cadherin、a-SMA表达水平的相关性；2）我们发现硒醇可以特异性打断Au-S键，构建了Au-S键偶联荧光染料的FRET探针，实现了乏氧条件下亚硒酸钠诱导肿瘤细胞凋亡过程中代谢物硒醇的检测，并为Au-Se键替代Au-S键构建新的纳米金应用平台奠定了基础；3）利用次溴酸特异性催化S=N的偶联，我们设计了一种HOBr存在下瞬时环化形成大共轭体系的探针(BPP)，极大地提高了检测的信噪比，实现了细胞及活体内本真HOBr超高灵敏、选择性检测，为进一步研究小分子调控胶原蛋白活性提供了实验依据；4）利用含Se-N键五元环的ebselen衍生物，构建了可逆响应GSH/H2O2水平变化的氧化还原动态平衡可视化探针，并进一步发现凋亡过程中的肿瘤细胞提高其还原性分子水平凋亡细胞出现逆向转化现象，此结果为讨论肿瘤复发具有一定的临床意义；5）我们发现超高活性的硒化氢可以特异性打断Se=N双键，利用苯并硒二唑作为报告基团，成功发展出一种超灵敏、特异性检测乏氧条件下亚硒酸钠诱导肿瘤细胞凋亡过程中信号分子H2Se的探针，并且发现该过程中亚硒酸钠诱导肿瘤细胞凋亡机制是非氧化胁迫。总之，在该基金资助下，发表第一作者及通讯作者SCI 论文14篇，影响因子均5.0以上。研究成果获省自然科学二等奖1项。培养硕士毕业生7人，在读博士生3人。参加国内学术会议6次。圆满完成项目预定研究目标和任务。