丹磺酰分子影像探针的构建及其在细胞凋亡成像中的应用

Molecular probes are the major driving force of molecular imaging science, providing many useful informations for the basic, preclinic and clinic research. Apoptosis (programmed cell death) plays a crucial role in the pathogenesis of many disorders, thus the detection of apoptotic cells can provide the physician with important information to further therapeutic strategies and would substantially advance patient care. Our previous work and other published results have demonstrated that dansyl probes can be used as imaging agents to specifically and selectively detect apoptotic cancer cells, but due to its limited light penetration depth associated with strong scattering for biological samples, the in vivo imaging are lack of high resolution. Rare-earth upconversion nanophosphors (UCNPs) show many attractive chemical and optical features, such as sharp emission lines, long lifetimes, superior photostability and no blinking. Such unique features are applied as the signal part for the design molecular probes by the conjugation of UCNPs and targets for both in vitro and in vivo imaging of apoptosis in this project. To further improve penetration depth and obtain higher targeted cell-specific probe for in vivo imaging, a series of UCNPs and targeted molecule (DNSBA derivatives and DNSBA-DEVD) are designed and synthesized as the potential probe for apoptosis imaging. The relationship between the structure of Dansyl-UNCPs from various approaches and related biological activities will be investigated using the established platform. After the biological activity screening, their specificities, sensitiveness and selectiveness of selected dansyl probes for in vivo imaging will be examed with the optical imaging and MRI in the apoptosis model in mice. Further mechanism experiments are explored to understand why and how the dansyl probe binds to apoptotic cancer cell. The rusults of this project will open an avenue for future in vivo experiments and for novel, self-renovation probes to detect apoptosis. It will also provide a new tool for apoptosis imaging with the same probe but for both in vitro and in vivo experiments.

寻找和研制性能优良的分子探针是分子影像学研究中的难点和技术瓶颈。本项目利用稀土上转换发光材料的高穿透性、光磁复合性、高化学稳定性等独特优点，针对前期研制的靶向细胞凋亡的小分子荧光探针穿透性差和生物背景干扰等不足，以稀土上转换纳米发光材料为信号组件，构建具有靶向凋亡的丹磺酰稀土上转换分子影像探针；利用我们建立的肿瘤细胞凋亡模型进行生物活性评价，明确适合于靶向成像的稀土上转换分子探针的结构；依托前期构建的整体动物模型和光学、磁共振成像技术平台，探索和比较不同结构的上转换分子探针在活体成像时的靶向性、特异性和敏感性，以期筛选出具有高灵敏度和特异性的分子影像探针；并开展深入的分子生物学研究，明确其靶向凋亡的作用机制。本项目是在我们已有研究工作基础上的延伸和深入，不仅将获得具有自主知识产权的靶向分子探针，而且将实现体内、体外利用同一分子探针对细胞凋亡的实时检测，并为活体凋亡成像提供敏感快速的新方法。

寻找和研制性能优良的分子探针是分子影像学研究中的难点和技术瓶颈，开发具有高灵敏性和特异性的分子影像探针用于检测细胞凋亡对疾病的诊断和治疗具有重要意义，可为疾病的早期检测、预警、诊断和药物疗效评估提供新的方法和工具。本项目利用稀土上转换发光材料的高穿透性、光磁复合性、高化学稳定性等独特优点，构建了多个新颖结构的分子探针，采用核磁共振、质谱等手段进行了结构表征和确证；搭建了细胞凋亡体内外评价平台，建立了多种细胞凋亡模型；开展了探针的生物活性评价、凋亡成像和特异性分子识别的研究；发现了具有自主知识产权的多个新型靶向凋亡的分子探针和特异性识别离子、信号分子和生物大分子的探针；通过深入的分子生物学研究，明确了丹磺酰分子探针靶向凋亡的作用机制，回答了丹磺酰分子探针能灵敏有效地检测凋亡细胞及其凋亡通路的特异性。本项目的完成，发现了具有多个自主知识产权的新型细胞凋亡分子影像探针，完成了其设计、制备、路线优化、生物学评价等研究。本课题紧跟分子影像学和细胞凋亡的热点研究领域，探索了如何解决实现建立检测细胞凋亡的分子影像探针的应用基础问题。此外，我们还基于稀土上转换纳米材料和细胞凋亡，系统性的总结了近年来的国内外科研进展与前沿；构建了多种重要的稀土上转换纳米材料和小分子探针材料，实现了其特异性的分子识别、成像、治疗以及诊疗一体化应用。.目前，我们如期超额地完成了该课题的预期研究内容和研究目标。迄今为止，我们在国际学术期刊上发表了署名本项NSFC资助的SCI论文28篇，申请国家发明专利2项，主编国际学术专辑Molecular Imaging of Protein and Peptides 1部，参编国际学术专著1部，在国际和全国学术会议上作大会报告、专题报告6次。在本项目研究成果的基础上，本课题组曾文彬教授作为项目主持人相继获得国家自然科学基金面上项目、湖南省自然科学杰出青年基金等多项课题。获得湖南省科技一等奖1项。