用于细胞与活体中超氧阴离子实时、原位动态监测的新型基因编码荧光探针的制备及应用

In cells, various free radicals are generated in the course of biological metabolism, while the most common ones are reactive oxygen species (ROS). A massive study shows that the genesis and development of many major diseases including tumor is closely related with the ROS level in cells. To achieve in situ real-time measurements of dynamic changes in superoxide levels, we introduced a “aldehyde tag” on the enhanced green fluorescent protein (EGFP) by gene clone technology, which could bioorthogonal react with aminooxy. Through bioorthogonal reaction, a novel genetically-encoded fluorescent probe is assembled, which could detect superoxide anion, the most important and the precursor of other ROS, in cells by chemiluminescence reaction. This probe not only inherits all advantages of chemiluminescence probe, including emission without excitation light, low background, high sensitive and instantaneous response, but also has lower toxic effect and higher quantum yield to achieve single-cell imaging and tissue depth imaging analysis. Since EGFP protein continuously expresses in cells, this probe could detect the level of ROS long-termly in tissue without excitation light, and simultaneously visualize the real-time changing of ROS level in situ while the process of tumorigenesis. The content of this project is a new strategy and pathway of free radical quantitative precision analysis.

细胞在新陈代谢过程中，会产生各种微量的自由基，其中活性氧自由基是机体内最常见的自由基。大量的研究证据表明，癌症等许多重大疾病的发生发展与细胞内活性氧水平的改变密切相关。为解决细胞、活体内自由基实时、原位、长时间的过程监测，本项目提出利用醛基与氧氨基在生物体内可发生生物正交反应这一特点，对增强型绿色荧光蛋白（EGFP）进行基因修饰，制备可对ROS中重要的也是其它活性氧生成的前驱体-超氧阴离子自由基发生化学发光反应的新型基因编码的荧光探针。该探针在融合了化学发光探针的无需激发光源、低背景、高灵敏度、瞬时检测等优点外，还具有更低的细胞毒性，更高的量子产率，可以实现单细胞成像及组织深度成像。由于EGFP在细胞中可以持续表达，此探针便于无激发光源长期检测组织中活性氧的变化，可以实现对肿瘤形成过程中活性氧水平变化的实时原位检测。项目提出也是自由基生物定量精准分析的新策略与新途径。

利用化学发光背景低，灵敏度高等特点，我们成功制备了化学发光探针EGFP-CLA，并初步检测了其发光性质。其次，利用小分子荧光探针毒性小，渗透性强，荧光性质稳定等优势，结合在炎症和肿瘤细胞中过量表达的白三烯A4水解酶的特异性底物，我们设计合成了一系列小分子荧光探针，用于细胞及活体内白三烯A4水解酶的活性检测。本课题选择了具有双光子性质的香豆素作为荧光团与白三烯A4水解酶的底物Aspbzl通过酰胺键结合，当在细胞中检测到白三烯A4水解酶时，酰胺键断裂，香豆素发出较强的荧光。基于双光子的较深的穿透深度的优势，我们实现了小鼠肺炎模型的无损成像检测，该探针有望应用于未来临床肺炎检测。此外，我们开展了绿色荧光蛋白与聚合物结合，共同检测金属离子钙离子的相关工作。绿色荧光蛋白具有无毒性，兼容性好等优势，结合聚合物的信号放大性质，可以较好的对细胞内的钙离子进行成像。绿色荧光蛋白的发射光谱与聚合物的吸收光谱具有一定的重叠，通过比率成像，可以实现钙离子的定量检测。