线粒体中锌离子和ATP动态平衡超分辨荧光成像及在前列腺癌早期诊断中的应用

The early detection of prostate cancer is a life-saving event in patients harboring aggressive disease. Diagnostic method based on prostate specific antigen (PSA) screening lacks specificity. Zinc ion as a marker for prostate cancer has been confirmed by numerous studies, reducing the concentration of zinc ions and ATP in mitochondria, associated with the inability of cancer cells to accumulate zinc ions. In contrast, prostate tissues of benign prostatic hyperplasia and prostate inflammation still have the ability to accumulate zinc ions, containing a similar level of zinc ions with that of healthy tissues. Therefore, endogenous zinc ions and ATP concentrations can be imaged as a biomarker for prostate cancer early detection and progression monitoring. However, the reported fluorescent probes imaged zinc ions and ATP with confocal microscopy, whose resolution was limited by diffraction to be 300 nm. Then it’s difficult to localize mitochondria with precision and map the distribution of Zind and ATP within mitochondria. In this project, applicants will introduce reported receptors whit specificities for zinc and ATP into the new type of xanthene fluorophores to construct ratiometric fluorescent probes for super-resolution fluorescence imaging of zinc and ATP, associated with the mitochondrial targeting ability. The probes will help to build the connections between homoestasis of zinc ions and ATP Gleason scores, which will be ultimately applied in an animal model for in vivo imaging, providing an effective reagent for the early detection of prostate cancer.

锌离子作为前列腺癌早期诊断标志物特异性优于临床采用的前列腺特异性抗原(PSA)，特征表现为线粒体中锌离子浓度降低先于前列腺癌发生，并在癌变过程中持续下降和引起线粒体ATP浓度升高，而前列腺增生和炎症组织细胞线粒体中具有和健康前列腺类似的锌离子和ATP水平，因此通过检测线粒体中锌离子和ATP浓度变化能够对前列腺癌做出早期诊断。已报道线粒体锌离子和ATP荧光探针存在分辨率低和线粒体定位不稳定的问题，未能分辨线粒体内部锌离子和ATP三维分布，对线粒体内锌离子含量测定误差大，没能建立线粒体锌离子浓度与前列腺癌发展的关系。本项目基于申请人在锌离子、ATP荧光探针和超分辨荧光染料和成像方面的进展，对超分辨荧光染料功能化，创制一系列SNAP蛋白标签线粒体定位的锌离子和ATP荧光探针，开展线粒体中锌离子和ATP动态平衡的超分辨荧光成像研究，并应用于前列腺癌早期诊断。

本项目发展超分辨荧光探针，具有超分辨成像的时空分辨率，在此基础上开展线粒体定位的锌离子和ATP的时空浓度荧光成像追踪，获得锌离子和ATP在线粒体中的动态分布规律。将获得的结果和数据分析用于多种前列腺癌细胞检测中，建立线粒体中锌离子和ATP浓度与格里森分数之间的关系，并最终用于前列腺癌模型动物活体成像。在项目执行期间，我们进行了荧光染料构效关系和发光机理研究、超分辨成像自闪荧光染料、锌离子探针定位识别、细胞器时空超分辨四维荧光成像、和荧光识别信息处理方法等方面的工作。突破了目前20 nm空间分辨率的瓶颈和难以动态成像的难题，获得了在9 nm/20 ms时空分辨率下连续超分辨成像40分钟等重要进展。实现了在线粒体、溶酶体、脂滴、细胞膜等多个细胞器中的精准定位和对内部锌离子等功能分子的动态识别。实现了在单分子定位下锌离子和质子的荧光传感，并分析获得了全细胞溶酶体内锌离子和质子的时空分布规律。通过单分子定位下发光的环境敏感性，建立了锌离子与线粒体、溶酶体等多个细胞器内部环境的依存关系。构建四维超分辨影像组学对包括前列腺癌在内的生理病理进行诊断和分型。共发表SCI论文66篇，其中包括1篇Chem. Soc. Rev., 2篇Coord. Chem. Rev., 2篇JACS，10篇Angew，1篇Chem. Sci., 3篇Chem. Commun.等。申请专利65项，授权专利72项，在资助期限内所申请专利获得授权的4项。