在单细胞水平超声和靶向微泡转基因的动态过程研究

When excited by ultrasound, microbubbles can temporarily and reversibly change cell membrane permeability, opening new opportunities for drug and gene delivery. However, ultrasound and microbubble mediated gene delivery currently has relatively low efficiency, which is mainly due to the insufficient understanding of the underlying mechanisms and the process, especially at single-cell level. To investigate the inherently dynamic and transient process of ultrasound and microbubble mediated gene delivery, well controlled experimental conditions and real-time monitoring and assessment methods are required. In this research project, targeted microbubbles will be employed. The selective attachment between targeted microbubbles and the target cells enables more precise targeting ability and better controlled delivery outcomes...Multiple experimental techniques, such as real-time spinning disk confocal fluorescent microscopy and patch clamp, combined with mathematic modeling and numerical simulation will be carried out to quantitatively investigate the dynamic trans-membrane process of genetic materials driven by ultrasound and targeted microbubbles, the physical characteristics of membrane ruptures or pores through which genetic materials transport across cell membrane and the kinetics of genetic materials inside the cells. Moreover, the influences of different ultrasound modes, ultrasound parameters, the physical properties of targeted microbubbles and their physical behavior stimulated by ultrasound to the dynamic process of gene transfection will be thoroughly studied...The aim of this research project is to unveil the dynamic process of genetic materials delivery mediated by ultrasound and targeted microbubbles in a spatial-temporally correlated manner, and the determinant roles of ultrasound conditions and microbubble dynamics in this process. Our research outcomes will help to develop ultrasound and targeted microbubble mediated gene delivery technique as a robust, reliable, and high efficient approach for applications that require controlled cellular delivery of genetic materials.

被超声激发的微泡可使其周围细胞的细胞膜通透性发生瞬态的、可逆的改变，从而为药物和基因的导入提供了新的方法。然而目前该技术的转基因效率相对较低，这主要是由于对于其在单细胞水平的发生机理的认识尚不充分。超声和微泡介导的转基因过程是一个不断动态变化的过程，因此只有在高度可控的实验条件下，采用实时的研究方法才能深刻揭示其发生机理。本项目将使用靶向微泡，其与目标细胞空间上的紧密连接，提高了微泡对细胞膜作用的可控性。本项目将在单细胞水平，综合运用实时共聚焦荧光显微成像技术、膜片钳等多种实时研究技术，结合数学建模和数值模拟等分析方法，探索超声和靶向微泡介导的基因导入的方式和过程、成功导入的条件以及基因在细胞内的动力学表现。在此基础上，研究超声激励方式、参数以及靶向微泡的物理特性及其超声响应对转基因过程的影响。本项目的研究成果将为完整地、时空连续地揭示超声和微泡转基因的微观动态过程做出原创性贡献。

超声和微泡的联合使用为靶向药物和基因导入提供了新的方法。该技术的临床应用前景广泛，国内外都有该技术进入临床实验的报道。然而目前该技术实现的基因转染效率相对较低，这主要是由于对其在单细胞水平的发生机理认识尚不充分。.在本项研究中，我们使用了靶向微泡，其与目标细胞的紧密连接，提高了微泡对细胞膜作用的可控性。我们综合运用了多种实时研究技术，结合数学建模和数值模拟等分析方法，在单细胞水平，深入研究了，超声和靶向微泡介导的质粒DNA通过细胞膜进入到细胞内的动态过程、过膜机制、成功导入的条件以及基因在细胞内的动力学表现等。.我们的研究结果表明超声和靶向微泡介导的质粒DNA的细胞导入是一个快速的、全细胞尺度的、激发多种导入机制的动态过程；并且导入过程是由超声模式所决定的。我们的研究成果揭示了超声和微泡实现基因导入的机制、动态过程和影响因素，为优化基于超声和微泡的靶向给药技术提供了理论基础，有助于推进该技术的临床转化。