胆固醇是细胞膜中重要的组成部分之一，其在细胞内的流动紊乱将导致一系列心血管及精神类疾病的发生。精准测定活细胞膜中胆固醇分子的跨膜转运过程，对理解胆固醇在细胞内的运动和代谢至关重要。目前测量方法多采用胆固醇类似物和标记胆固醇,已被证明其运动特征与本体胆固醇存在差异。为了实现对细胞膜中本体胆固醇的快速跨膜过程进行精准测定，本项目将基于申请人在单细胞胆固醇电化学测量多年的研究积累，建立快速电致化学发光分析新体系，将电化学传感的时间分辨率提升到亚微秒级，从而可直接测量本体胆固醇在单个活细胞膜中跨膜转运速度。研究工作将在磷脂双分子层模型和单个活哺乳动物细胞两个层面开展，彼此验证，以期获取胆固醇分子跨膜转运的精确速度。在此基础上，对胆固醇跨膜转运的相关膜蛋白含量进行调控，研究膜蛋白在胆固醇跨膜转运中的作用以及可能的分子机制，最终为胆固醇转运相关疾病的病理研究提供有效的技术平台和生物学信息。

Cholesterol is one of the main components in cellular plasma membrane, and the dysfunction of cholesterol trafficking inside the cells has been proved to induce multiple diseases, including arteriosclerosis and the psychiatric diseases. Therefore, the accurate measurement of trans-membrane transport (flip-flop) of cholesterol molecules in plasma membrane at single living cells is essential to understand the trafficking and metabolism of cholesterol in cell. At present, cholesterol analogues and labeled cholesterol are used in the measurement of flip-flop rate, which have been proved to alter the intrinsic physical and chemical properties of cholesterol in cell membrane. Based on our previous research of single cell electrochemiluminescence (ECL) cholesterol measurement, this project will aim at the development of a fast ECL measurement method with a temporal resolution of sub-microseconds, to directly measure flip-flop rate of native cholesterol in plasma membrane at single living cells. The time of flip-flop rate of native cholesterol in the lipid bilayer and plasma membrane at single living mammalian will be measured. Moreover, the function and molecular mechanism of transport protein in plasma membrane involved in the flip-flop of cholesterol will be investigated. The success in this project will provide a technical platform and biological information for the research on the pathology of cholesterol transport related diseases.