基于高性能遗传编码荧光探针的中心碳代谢活细胞成像与时空动态分析

The Central Carbon Metabolism pathway is vital for cellular metabolic activity, and closely related to various diseases that threaten human health, e.g. diabetes and cancer. Genetically encoded fluorescent sensors have excellent specificity and spatiotemporal resolution, and are well suited for analysis of the production, transport and transformation of metabolites in live cells and in vivo. However, these sensors are currently availabe with limited species and colors, which can not fullfill the needs for systematic analysis of cellular central carbon metabolism. In this research project, we intend to innovate the principles sensor construction¸ and systematically develop a series novel, high performing, multi-color and differentially-locating fluorescent sensors for the core metabolites in the Central Carbon Metabolism. Based on these sensors, we aim to implement real time, high spatiotemperal resolution, multi-parameter imaging of cellular central metabolism in single cell level, subcellular level and in vivo. We will further analysis the dynamics and regulations of the production, transport and transformation of these core metabolites, using cancer cells and tumor microenvironments as model systems. These studies will provide toolkit of fluorescent sensors and imaging methodologies for the study of central carbon metabolism, which is not only useful for the understanding of spatiotemporal dynamics and regulation of cell metabolic networks, but also provides new approaches for the diagnosis and drug discovery of diseases associated with metabolism imbalance.

中心碳代谢是细胞代谢活动的核心，与威胁人类健康的糖尿病、癌症等各类疾病密切相关。遗传编码荧光探针具有优异的特异性和时空分辨率，适于在活细胞与活体内分析代谢物的产生、运输和转化。但现有高性能代谢物探针种类与颜色有限，难以满足系统研究细胞中心代谢的需求。本项目拟发展基于新原理的遗传编码探针构建方法，针对细胞中心碳代谢途径的多种核心代谢物系统开发不同颜色与亚细胞结构定位的新型高性能遗传编码荧光探针，实现单细胞与亚细胞水平、实时动态、高分辨率、多参数的细胞中心碳代谢成像，并以肿瘤细胞与肿瘤微环境为例分析这些重要代谢物质在细胞与机体内的产生、运输和转化动态过程与调控规律。本项目研究将形成针对细胞中心碳代谢的荧光探针与代谢时空动态分析技术工具箱，不仅可为人们在系统解析细胞代谢网络的时空动态变化与调控规律提供新技术与新方法，也可为疾病的诊断与药物发现研究提供新的手段。

中心碳代谢是细胞代谢活动的核心，与威胁人类健康的糖尿病、癌症等各类疾病密切相关。遗传编码荧光探针具有优异的特异性和时空分辨率，适于在活细胞与活体内分析代谢物的产生、运输和转化。但现有高性能代谢物探针种类与颜色有限，难以满足系统研究细胞中心代谢的需求。本项目发展了荧光RNA等基于新原理的遗传编码探针构建方法，针对细胞中心碳代谢途径的多种辅酶、羧酸、氨基酸等核心代谢物系统开发了二十余种不同颜色与亚细胞结构定位的新型高性能遗传编码荧光探针，实现单细胞与亚细胞水平、实时动态、高分辨率、多参数的细胞中心碳代谢成像。在细胞代谢控制方面，本项目在光遗传学技术上取得了突破，发展了多种RNA核心代谢过程的光控系统，实现了RNA转录、剪切、降解和翻译的定时、定量精密调控。项目还以肿瘤细胞与肿瘤微环境为例分析了重要代谢物质在细胞与机体内的产生、运输和转化动态过程与调控规律。本项目研究形成了 针对细胞中心碳代谢的荧光探针与代谢时空动态分析技术工具箱，不仅可为人们在系统解析细胞代谢网络的时空动态变化与调控规律提供新技术与新方法，也可为疾病的诊断与药物发现研究提供新的手段。