新型AFM-光学复合成像系统的研制及其在心肌细胞力学性质和药物筛选的应用

The study of mechanical properties of cardiomyocytes is critical to undertand the fundamental mechanism of cardiopathy. Scientists including myself have used the atomic force microscopy (AFM) and other nanotechnologies to measure the mechanical property including contraction and stiffness of carcdiomyocytes effectively. However, these technologies have the limits such as touching and disturbing cell surface, measuring only local areas and so on. Recently several 3D imaging techniques have been developed to measure the structure and molecular motion in cells, but these 3D imaging techniques can actually be used to measure the contraction of cardiomyocytes without touching cells and overcome the limits of current techniques. This proposal is to develop a new imaging technique by combining the AFM and 3D imaging techniques. Also it will couple the AFM and calcium imaging in order to measure the correlation between cardiomyocyte contraction and its calcium flux that is also an important property of cardiomyocytes. The change of mechanical property of cardiomyocytes during the development of an embryo is an important but not well studied field. We will prepare the cardiomyocyte model that can represent the phenotype of real cardiomyocytes at varying stages of the embryonic development by using the iPSC technique, and then apply our new imaging technique to measure the change of mechanical properties of the cardiomyocyte model. We will also try to use the proposed technique to develop a platform of drug screen for cardiopathy.

研究心肌细胞力学性质对于深入认识心脏病生理病理机制进而发展出心脏病新疗法具有重要意义。包括申请人在内的科学家应用原子力显微镜（AFM）以及其他纳米技术，有效测量了心肌细胞的收缩和硬度等力学性质，但现有技术仍然有接触扰动细胞，或者仅测量局部区域等局限性。近年来为了观测细胞内结构和分子运动发展出了几种卓越的三维光学成像技术，而这些三维成像技术其实可以非接触地精确测量心肌细胞的整体收缩，能有效弥补上述局限性。本课题计划建立将AFM和三维光学成像耦合的新型复合成像系统，并在系统中引入结合AFM和钙离子成像的新技术使之能够测量钙离子流和心肌细胞收缩关联性这一重要性质。胚胎发育过程中心肌细胞的力学性质变化是个重要而研究很少的领域，本课题计划应用诱导干细胞技术培育能表征不同发育阶段的心肌细胞模型，并使用此复合成像系统研究细胞的力学性质变化。另外还计划将该复合成像系统应用于心脏病药物筛选的研究。

研究分子和细胞层次的异质性是理解生物个体异质性这一基本生命现象的基础。相对于传统的系综测量技术，单分子荧光成像技术，因为能避免系综平均，在生理条件下对单个的细胞或分子直接进行观测，所以对于研究生物分子和细胞的异质性有突出的优越性。在本课题中，我们建立了多波长多模式的三维单分子荧光成像及钙离子成像相耦合的新型复合成像系统，并用于研究心肌收缩过程中的单细胞异质性和GlnBP及4.1G等重要蛋白分子的构象和功能异质性。我们发现心肌细胞收缩和钙离子释放之间的同步，并且发现肥大性心肌病（HCM）细胞在发育早期即表现出异常收缩特征, 这表明具有遗传缺陷的心肌细胞在发育较早期已经开始表现出遗传疾病的表型，但是实际心脏器官却是在生物体成熟很长时间后才发生病变，这个差异说明可能有其他的机制能够调控心肌细胞的异常收缩。在蛋白分子异质性方面，我们观测到了谷氨酰胺结合蛋白GlnBP多重构象结构，表明构象选择机制在其和配体的相互作用中发挥重要作用。在天然无序蛋白4.1G和配体结合的单分子实验中，我们观测到了4.1G的多重构象以及配体结合对构象的调控，结合计算和其他观测技术，我们发现多重构象有助于提高结合效率，这也许暗示获得构象多样性是生物大分子在进化中提高活性的一个普遍策略。我们的研究对于揭示心肌疾病机制、蛋白作用机制以及药物研发等方面都有重要意义