细胞核靶向次氯酸荧光探针的构建及其在生物成像中的应用

Hypochlorous acid is one of the important reactive oxygen species in physiological systems. Low level of hypochlorous acid plays essential roles in anti-inflammation and immune processes. However, elevated level of hypochlorous acid would inevitably bring oxidative damages and lead to many diseases. Nucleus contains almost all of the genetic materials, and the entry into nucleus of hypochlorous acid can lead to gene damages and many serious diseases such as cancer. Consequently, detection of hypochlorous acid in nucleus is of great significance for the investigation of the interactions between nucleus and hypochlorous acid as well as the pathological studies of related diseases. Considering the deficiency of fluorescent probes for nuclear hypochlorous acid, in this project, we for the first time present the construction of fluorescence probes for the detection of nuclear hypochlorous acid. The nucleus targeted moiety is obtained from the analysis of targeting mechanisms and the exploration of the relationship between molecular structure and nuclear selectivity. The HClO-responsive moiety with high selectivity and sensitivity is designed on the basis of the fluorescence dyes with high quantum yield, including fluorescein and naphthalimide. Fluorescent probes for nuclear hypochlorous acid can be constructed by the reasonable combination of the two kinds of moieties. These probes designed in this project would be powerful biotools for the investigation of interactions between HClO and nucleus, pathological studies, and the diagnosis of relative diseases.

次氯酸是生命体系内重要的活性氧簇化合物之一，低浓度次氯酸在免疫和炎症反应中起重要作用，而高浓度次氯酸会带来机体损伤，导致诸多疾病。细胞核存储着几乎所有的遗传物质，次氯酸侵入细胞核将导致基因损伤，诱发癌症等重大疾病。因此，检测细胞核内次氯酸对研究次氯酸与细胞核相互作用及相关病理至关重要。针对目前缺乏细胞核靶向次氯酸探针这一问题，在本项目中，我们首次提出构建专一靶向细胞核、同时选择性响应次氯酸的荧光探针。本项目拟通过分析小分子靶向细胞核的内在机制，探索分子结构对细胞核靶向性的影响，得到专一靶向细胞核的模块。利用高量子产率的荧光素和萘酰亚胺等染料作平台，设计高选择性和灵敏度的次氯酸响应模块。合理整合细胞核靶向模块和次氯酸响应模块，构建靶向细胞核的次氯酸荧光探针。本项目拟研制的细胞核靶向次氯酸探针可为研究次氯酸与细胞核相互作用提供有力工具，在病理学研究和相关疾病诊断中具有重要意义。

细胞内髓过氧化氢酶可催化过氧化氢生成具有强氧化性的次氯酸。细胞内的次氯酸产生于炎症反应和免疫反应，主要用于快速有效的杀灭病原体。然而由于次氯酸氧化性极强，它在消灭病菌的同时会带来机体损伤，导致诸多疾病。细胞核存储着几乎所有的遗传物质，次氯酸侵入细胞核将导致基因损伤，诱发癌症等重大疾病。因此，检测细胞核内次氯酸对研究次氯酸与细胞核相互作用及相关病理至关重要。在本项目中，申请人首先探索并筛选了能够靶向核酸的荧光平台，并在平台基础上修饰了次氯酸响应位点，最终得到3例能够检测细胞核、核仁中次氯酸的荧光探针。利用此3例探针，我们确认了外源性次氯酸可以抵达活细胞核，鱼藤酮毒素可以诱导细胞核内次氯酸浓度升高，并探究了巨噬细胞在炎症模型下细胞核内次氯酸含量的变化。此外，基于在本项目中对分子靶向性的研究结果，申请人进行了拓展与延伸，研制了三类性能优异的荧光探针：构建出双色可逆监测细胞凋亡过程的荧光探针；设计合成了双色检测细胞自噬过程的荧光探针；研制了双色区分成像不同细胞器或亚细胞结构的荧光探针。申请人利用细胞凋亡荧光探针，观测了细胞健康状态的可逆变化，探究了维生素C对鱼藤酮毒素的解毒作用。利用细胞自噬的荧光探针，监测了细胞自噬过程中细胞对溶酶体、细胞质中pH值的调控作用，双色成像了线粒体自噬过程，探究了不同浓度活性氧对线粒体自噬过程的影响。利用双色双靶向型荧光探针，探究了不同细胞器之间的相互作用和内在联系，探究了高脂环境、饥饿环境下线粒体与脂滴的接触行为，成像了内质网膜上的相分离状态，确认了在胆固醇、棕榈酸诱导下内质网内可形成筏区，内质网氧化应激过程中，会形成大块筏区。我们研制的荧光探针可以作为重要分子工具推动生物医药领域的发展，我们利用探针进行生物先导性实验得到的重要结论，可为生物学的发展提供实验和理论基础。