单一微泡的声致细胞膜穿孔效应机制研究

发布时间：2018-03-11 16:21

本文选题：飞秒脉冲激光　切入点：微气泡　出处：《上海交通大学》2010年博士论文　论文类型：学位论文

【摘要】： 超声技术已经被证明能够暂时性的增强细胞膜的渗透性。在超声对比度增强剂如微气泡的辅助下,超声能量在细胞膜上通过机械作用在细胞膜上造成一定的损伤或者穿孔,从而提高细胞质膜的渗透率,使得原来不可能渗透过细胞膜的治疗物质,如:治疗药物、基因、蛋白质及其它生物大分子等通过细胞膜上的穿孔运输至细胞内,这一过程被称为声致穿孔效应。由于没有合适的控制及实时监测技术,细胞水平上的单一微气泡的声致穿孔效应对细胞膜的渗透率暂时性增强机制还不是很清楚。传统研究声致穿孔效应的方法有以下3个缺点:1、不能实现真正意义上的可控的单一气泡与细胞相互作用;2,不能实时地对声致穿孔效应的动态过程进行观察;3、无法实时测量细胞对声致穿孔效应的反应。本研究的研究内容主要包含2个方面:1、微米尺度的单一微气泡的非接触式三维操控; 2、可控的单一微气泡的声致穿孔效应对细胞膜的渗透率暂时性增强机制。在本课题中我们使用高速光学摄像技术及高频超声成像技术,研究了高能飞秒脉冲激光在水中自聚焦发生光学诱导击穿、产生并捕获微气泡的动态过程。我们还应用超声技术定量研究了飞秒脉冲激光束对捕获的微气泡的横向束缚力。通过光学技术与超声技术的结合实现了微米尺度的单一微气泡的非接触式三维操控。我们用能量较低的超声波将被捕获在光束内的微气泡放置到靶向细胞附近指定的位置(控制精度达到微米量级)。在高能超声的作用下,微气泡剧烈收缩、膨胀最终崩灭,与细胞发生相互作用在细胞膜上产生穿孔从而使细胞膜的渗透性增强。我们采用了四种独立的实时监测手段对这一过程进行了监测:1)M-mode超声成像;2)B-mode超声成像;3)高速光学摄像技术;4)电压钳技术。我们研究了各种物理参数对声致穿孔效应的影响,包括:1)超声能量;2)脉冲重复频率PRF (Pulse repetition frequency); 3)超声脉冲数量;4)微气泡与细胞壁的间距等。通过本课题的研究,取得的主要成果如下:1)阐述了自聚焦飞秒脉冲激光在水中产生并捕获微气泡的动态过程;2)应用飞秒脉冲激光及超声技术,首次实现了微气泡非接触式三维操控,弥补了传统光镊技术的不足;3)分析在单一细胞水平上,声致穿孔效应与空穴效应之间的联系;4)定量分析微气泡通过声孔作用于细胞膜发生作用的有效距离;5)确定了声致穿孔效应的动态过程并确定其最优化参数。
[Abstract]:Ultrasound technology has been shown to temporarily enhance cell membrane permeability. With the aid of ultrasound contrast enhancers such as microbubbles, ultrasonic energy causes certain damage or perforation to the cell membrane through mechanical action on the cell membrane. Thus increasing the permeability of the cytoplasmic membrane, making it impossible to penetrate the membrane of the treatment substances, such as therapeutic drugs, genes, proteins and other biological macromolecules through the perforation of the cell membrane into the cell. This process is called acoustic perforation effect. In the absence of proper control and real-time monitoring technology, The effect of acoustical perforation of a single microbubble at the cellular level on the mechanism of transient enhancement of cell membrane permeability is not well understood. The traditional method of studying acoustic-induced perforation has the following three disadvantages: 1, which cannot be realized in real sense. Can not observe the dynamic process of acousto-induced perforation in real time, and can not measure the response of cells to acousto-induced perforation in real time. The main contents of this study include two aspects: 1) the non-contact three-dimensional manipulation of a single micro-bubble on a micron scale; 2. The temporary enhancement mechanism of membrane permeability due to the sound-induced perforation effect of a controllable single micro-bubble. In this paper, we study the optical induced breakdown of high energy femtosecond pulse laser in water by using high speed optical camera and high frequency ultrasonic imaging. The transverse binding force of femtosecond pulse laser beam to the captured microbubble is also quantitatively studied by ultrasonic technique. The micron scale is realized by combining optical technology with ultrasonic technology. The non-contact three-dimensional manipulation of a single micro bubble. We use low-energy ultrasound to place microbubbles trapped in a beam of light at a certain location near the target cell (control accuracy is of a micron order of magnitude). Under the action of high-intensity ultrasound, the microbubble shrinks violently, and the expansion eventually collapses. Interaction with the cell causes perforation on the cell membrane to enhance the permeability of the cell membrane. We have used four independent real-time monitoring methods to monitor this process. The influence of various physical parameters on the acousto-induced perforation effect has been studied. These include: 1) ultrasonic energy 2) pulse repetition rate PRF pulse repetition frequency, 3) ultrasonic pulse number 4) the distance between microbubbles and cell walls, etc. The main achievements of this paper are as follows: 1) the dynamic process of self-focusing femtosecond pulse laser producing and capturing microbubbles in water is described. (2) femtosecond pulse laser and ultrasonic technology are used. For the first time, the non-contact three-dimensional manipulation of microbubbles is realized, which makes up for the deficiency of traditional optical tweezers. The relationship between acoustical perforation effect and hole effect) quantitative analysis of the effective distance from which the microbubble acts on the cell membrane through the acoustical hole 5) the dynamic process of the acousto-induced perforation effect is determined and its optimum parameters are determined.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R312

【相似文献】