溶酶体靶向的酸性磷酸酶荧光探针对自噬过程的影像学示踪

As a key protease for the autophagy process of lysosomes, the disorder of acid phosphatase activity is closely related to the major diseases, such as prostate cancers. However, it still lacks effective tools for real-time monitoring the activity of acid phosphatase in lysosome. The present research project is to design a novel lysosome-targetable fluorescent probe for responding to the activity of acid phosphatase and its sensing mechanism, achieving the real-time and in situ imaging analysis of the activity of acid phosphatase and the behavior of autophagy in lysosome. (1) Screening the cyclone ratine molecules with the ability of targeting lysosome, designing a molecular unit that can produce fluorescent responses to the activity of acid phosphatase, and synthesizing the lysosome-targetable fluorescent probe for the analysis of the activity of acid phosphatase. (2) Explaining the relationship between the efficiency of targeting lysosome and the molecular structures of different cyclones and the sensitive responding mechanism to the activity of acid phosphatase, and establishing in situ imaging method of the activity of acid phosphatase in living cells. (3) Imaging analysis of the activation process of acid phosphatase through the plasmid transfection and expression of the lysosomal membrane-associated protein, and revealing the relationship between its activity and the autophagy process. (4) Studying the difference of the acid phosphatase’ levels between normal cells and corresponding lesions, and establishing the relationship between the activity of acid phosphatase and the disease process of prostate cancers. The expected results of the project will provide the effective means for understanding lysosome-related physiological and pathological mechanisms.

酸性磷酸酶是溶酶体自噬过程的关键性蛋白酶，其活性失调与前列腺癌等重大疾病密切相关。然而，对溶酶体内酸性磷酸酶活性的实时监测仍然缺乏有效的手段。本项目拟探索溶酶体靶向及酸性磷酸酶活性响应的荧光探针及其传感原理，实现对溶酶体内酸性磷酸酶活性及其细胞自噬行为的实时影像学分析。（1）筛选具有溶酶体定位功能的轮环藤宁类分子，设计对酸性磷酸酶活性产生荧光响应的分子单元，从而合成出溶酶体靶向的酸性磷酸酶荧光探针；（2）阐明不同轮环结构定位溶酶体的效率以及对酸性磷酸酶活性响应的敏感机制，建立细胞原位酸性磷酸酶活性响应的光谱影像学方法；（3）通过溶酶体膜相关蛋白质粒的转染及表达，对酸性磷酸酶激活过程进行实时影像学分析，揭示其活性与自噬过程的关联性；（4）研究正常细胞与对应病变细胞内酸性磷酸酶水平的差异，建立酸性磷酸酶活性与前列腺癌病变过程的对应关系。预期成果将为理解溶酶体相关的生理病理机制提供有效手段。

当细胞坏死、凋亡以及一些疾病发生时，磷酸酶活性会被异常激活。因此，对磷酸酶活性的原位实时分析，不仅能够阐述其与细胞自噬过程的关联性，而且能够帮助理解溶酶体相关的生理病理机制。针对这一问题，我们制备了同时对磷酸酶和信号分子产生荧光响应的单分子探针，实现了活细胞中硫化氢对磷酸酶活性调节的实时影像观测，发现细胞内硫化氢浓度的升高或者降低都会引起磷酸酶活性的降低，揭示了磷酸酶活性与硫化氢水平的相关性。研究结果对硫化氢在细胞信号传递以及磷酸酶相关各种人类疾病的理解具有十分重要的意义。与此同时，还构建了多响应、多定位的光学探针以及寡核苷酸类荧光探针，实现了活细胞内信号分子对蛋白/酶活性的调控以及遗传物质动力学的分子影像分析，及肿瘤的跨平台检测。相关研究成果在Angew. Chem. Int. Ed.、ACS Nano、Anal. Chem.等高影响力的国际期刊上发表论文21篇。