基于共聚焦显微成像的三维数字图像相关技术及在肿瘤细胞迁移侵袭定量表征中的应用

Based on the high-performance laser confocal microscope platform, on one hand, this project focus on fabricating extracellular matrix materials with biological compatibility, controllable adhesion ligand distribution and adjustable fluorescent particle density and establishing a biological experiment system to evaluate the capability of tumor cell migration and invasion process. On the other hand, with the high resolution laser confocal optical tomography images captured in experiment, fluorescent speckle deconvolution algorithm and speckle particle center localization algorithm are proposed to obtain the curved surface parameters in the deformed image with aid of global least-square fitting algorithm. After that, a novel three-dimensional digital image correlation technique is developed by introducing a new affine shape function with curved surface deformation control parameters, by which the three-dimensional displacement fields exerted by single cell on the extracellular matrix are quantitatively qualified and the spatiotemporal characteristics of the tumor cell migration and invasion process is studied. Besides, by means of some biological techniques, for instance, immunofluorescence staining, the effects of extracellular physicochemical microenvironment on the migration and invasion ability of tumor cell is evaluated, which provides evidence for exploration of single cell migration and invasion in biomechanics. In summary, the new project is capable of offering quantitative measurement techniques and reliable biophysics experimental supports for systematically studying the physiological and pathological mechanism of tumor cell migration and invasion.

本项目将依托高性能激光共聚焦显微镜平台，制备具有生物相容性、粘附配体分布空间可控且荧光粒子（人造散斑）密度可调的细胞外基质材料；搭建用于肿瘤细胞迁移侵袭研究的生物力学实验体系；针对拍摄的高分辨激光共聚焦光学断层扫描图像，建立专门适于荧光粒子散斑图像的高效粒子解卷积算法和精准的散斑粒子中心定位算法；通过粒子中心全局最小二乘拟合技术获取变形后基底图像曲面控制参数，引入包含子区曲面变形信息的仿射形函数，发展一整套全新的三维数字图像相关技术；精确定量细胞与胞外基质相互作用的三维位移场，探究肿瘤细胞迁移与初始侵袭的时空动态变化特性；结合免疫荧光染色等相关生物学实验技术，研究胞外理化微环境变化对于肿瘤细胞迁移侵袭能力的影响，从生物实验力学角度考察细胞侵袭的一般规律，为系统研究肿瘤细胞迁移侵袭的生理病理机制提供新的精准测试工具和可靠的生物物理实验数据。

力学因素在肿瘤迁移侵袭中起重要作用。项目制备了具有生物相容性、粘附配体分布空间可控且荧光粒子（人造散斑）密度可调的细胞外基质材料，搭建了用于肿瘤细胞迁移侵袭研究的生物力学实验体系，开发了新的数字图像相关算法，实现了高性能牵引力显微镜测量，研究了胞外理化微环境变化对于肿瘤细胞迁移侵袭能力的影响。发现转化生长因子(TGF-β)会协同基底刚度促进MDA-MB-231乳腺癌细胞迁移，且受细胞收缩力的调节；基底刚度是通过激活TGF-β前体来调节肝癌细胞TGF-β1 信号通路，且这种激活作用依赖Integrin介导产生的细胞收缩力；发现B淋巴细胞活化过程中免疫突触内会产生nN级别的牵引力，且受F-actin骨架重构，以及myosin IIA和dynein介导的细胞收缩的调控；发现ZnO纳米颗粒能显著地改变气道平滑肌细胞的基础和KCl刺激的收缩力，Clathrin介导的细胞内吞参与其中。这些结果为系统研究肿瘤细胞迁移侵袭的生理病理机制提供新的定量工具和可靠的细胞力学实验数据。