活细胞内机械应力传递的可视化研究

Mechanical stress transmission in cells plays an important role in the regulation of cell functions, but currently there is no related technology which can be used to directly observe the transfer of intracellular mechanical stress. Based upon the fluorescence resonance energy transfer (FRET) technology, the objective of this project is to construct a novel visual stress detection probe in living cells with a molecular spring and fluorescent protein using subcloning technology, which will be suitable for stress transfer detection in every part of any specific cellular structure, and achieve the visualization of intracellular mechanical stress transfer. This probe will be transfected into the cell, and a parallel plate flow chamber system and gel pulling system, which supply steady shear stress and stretch to stimulate the cell, will be restructured for real-time observation on FRET fluorescence microscope. Then the real-time mechanical stress transfer between cell membrane, cytoskeleton and focal adhesion in cell will be observed using FRET fluorescence imaging technology in living cell under external stress application. Through image processing and mathematical modelling and data fitting, the pathway and modes of intercellular mechanical stress transfer including corresponding adjustment mechanism will be analyzed, and the basic rule of mechanical change will be further investigated in molecular or cellular levels. This project will also provide new technology and ideas for the study of pathogenesis and prevention for stress related diseases.

机械应力在细胞内的传递在调节细胞功能变化过程中起着重要作用, 但目前尚无相关技术可以直接观测到胞内机械应力的传递。本项目拟在荧光共振能量转移技术（fluorescence resonance energy transfer，FRET)基础上，运用亚克隆技术，采用分子弹簧及荧光蛋白构建新型活细胞应力检测可视化探针，使其适合细胞各部分特定结构的应力传递检测，从而实现细胞内机械应力传递的可视化；运用此探针转入细胞，采用经改造适合于FRET的平行平板流动腔系统与基质胶牵拉系统，对培养细胞分别提供剪切应力和牵张力，利用FRET荧光图像实验技术，实时记录外界应力作用下机械应力在细胞膜-细胞骨架-局部粘着斑间进行传递的过程，通过图像处理与数学建模拟合分析胞内机械应力传递的途径和模式及相应的调节机制，深入探索细胞分子层次的力学基本变化规律及其科学本质，为应力相关疾病的发病机理和防治研究提供新的技术与思路。

机械应力在细胞内的非均匀性传递可能是细胞定向迁移等细胞功能变化过程的关键因素，然而该领域的研究进展缓慢，主要原因可能在于缺乏相关技术可以直接观测到胞内机械应力的传递。为了解决这一难题，本项目基于荧光蛋白的荧光共振能量转移（fluorescence resonance energy transfer，FRET) 技术，通过常规亚克隆技术，巧妙地采用分子弹簧及荧光蛋白设计并构建了一系列新型活细胞应力检测可视化探针，通过在探针两端添加特异性连接标签，使其适合细胞各部分特定结构的应力传递检测，从而实现了细胞膜张力、细胞膜脂阀至细胞骨架微丝、细胞骨架微丝至局部黏着斑、局部黏着斑内部Paxillin蛋白、以及神经细胞突触活性带区域机械应力改变的可视化检测；同时，设计构建了细胞定向迁移相关的HSP27蛋白自聚合、PIM蛋白活性变化的活细胞动态检测FRET探针；运用这些探针，系统地检测了在定向剪切应力作用下，机械应力在细胞膜-细胞骨架-局部粘着斑间进行传递的过程，并通过图像处理与数学建模拟合分析胞内机械应力传递的途径和模式及相应的调节机制，发现了定向剪切应力作用下对细胞极性改变与定向迁移的信号调控，首先发生在细胞膜至细胞骨架应力传递的层面而非细胞膜张力层面，这一过程受到细胞膜流动性与细胞骨架完整性的直接影响。本项目的研究成功解决了细胞内应力不可检测的难题，为力生物学领域的研究提供了一系列有力的工具；同时初步揭示了细胞内应力沿细胞膜-细胞骨架-局部粘着斑间依次传递，并引发细胞定向迁移的信号途径，深入探索了细胞分子层次的力学基本变化规律及其科学本质，也为应力相关疾病的发病机理和防治研究提供新的技术与思路。