单囊泡形成过程化学动力学的精准测量

Vesicle trafficking is an important biological process in cells. The formation of vesicles is the start of the vesicle trafficking process, and plays vital role in remaining the running cycle of vesicle trafficking. Better knowledge about the chemical process and kinetics of vesicle formation may promote our understanding on the function of vesicle trafficking process and the mechanism of vesicle-related disease. Unfortunately, limited by the measurement technology, it is still a challenge of directly monitor the kinetics of a single vesicle formation. This proposal aims at the chemical kinetics of single vesicle formation. We try to develop high tempo-spatial resolved spectroscopy and imaging methodology for dynamically monitoring the formation process of vesicles. We attempt to utilized TIRF optics, femtosecond laser excitation and pulse sequential control techniques, to over the challenges in monitoring the kinetics of life systems with small size, fast dynamics, multi-components and weak signals, through simultaneously detection of Mie scattering, fluorescence and Raman scattering photons enhanced by the plasmonic probes. Via the precision measurement on the kinetics of vesicle formation, we will develop reaction models for 1-2 types of typical vesicle formation, and further reveal the kinetics parameters, associated factors and the cellular response to the external stimuli.

囊泡转运是细胞内重要的生命过程。囊泡的形成既是囊泡转运过程的起点，也是保证囊泡转运过程循环进行的重要物质基础。认识囊泡形成的原初化学过程及动力学特性，对深入理解囊泡转运的功能及相关疾病的发生机制有重要的科学意义。受限于测量技术，目前仍无法直接测得单个囊泡形成的化学动力学。本项目拟围绕“单囊泡形成化学动力学”的科学问题，发展高时空分辨的光谱探测和分子成像方法，通过对Plasmon纳米探针增强的Mie散射、荧光及拉曼信号的协同探测，结合TIRF光路、飞秒激发及脉冲时序控制等关键技术，攻克小尺寸、快变化、多组分、弱信号生命体系中动力学测量的关键科学和技术难题，实现囊泡形成过程化学动力学的精准测量。争取建立1-2种典型囊泡形成过程的化学动力学模型，测得包被蛋白组装的动力学参数，从反应动力学角度精准描绘囊泡形成的化学过程。继而揭示囊泡形成的化学动力学参数、关联因素、以及细胞受激条件下的动力学响应。

本项目围绕囊泡形成的核心科学问题，旨在通过对囊泡形成动力学的精准测量，揭示囊泡形成的动态过程及其关联因素；重点是发展新的精准测量技术和方法，攻克小尺寸、快变化、多组分、弱信号生命体系中动力学测量的关键科学和技术难题。通过项目的执行，成功研制了多种高灵敏的纳米探针，包括纳米激光探针、荧光寿命探针、拉曼探针以及pH相应探针等；发展了多种光谱信号的检出方法，包括高通量信号提取、高空间分辨单颗粒定位、高时间分辨信号跟踪等；成功实现了单细胞中溶酶体囊泡形成过程的动态测量，解析了溶酶体囊泡中化学环境演化的动力学规律。项目研究期内发表相关研究论文14篇。申请发明专利3项，获得专利授权1项。培养博士后1名，博士研究生3名，硕士研究生3名。本项目以按计划圆满完成研究任务。