飞秒激光刺激技术对细胞功能调控的研究

发布时间：2018-11-26 11:20

【摘要】：生物光子学是光学与生物学高度交叉的前沿领域,飞秒激光技术的进步,为生物光子学提供了一系列新技术、新方法、新思路、新理论。目前基于飞秒激光的生物光子学已经在多光子成像、细胞显微操作等领域得到了深入的研究和广泛的应用。然而,此前飞秒激光刺激对细胞的影响和损伤机制的研究相对粗糙,飞秒激光脉冲和细胞相互作用的机制本身,以及细胞对飞秒激光刺激的应激反应等问题却一直尚未清晰阐明。在这样的大背景之下,本文研究了飞秒激光刺激对细胞过程的影响和调节机制,提出了新的飞秒激光调控细胞内分子行为的方法,并进一步发展了超快成像技术。本文首先综述了基于飞秒激光的生物光子学的进展、近年来研究的关键问题与发展现状。接着,作为相关生物分析的基础理论,简要介绍了钙离子的相关背景,包括钙离子的存储、生理作用、与之相关的分子信号等。进一步地,介绍了基于飞秒激光器、共聚焦显微镜、CCD等设备构成的显微操作系统,并讨论了在此系统之上使用飞秒激光调控细胞内钙离子浓度变化的实验现象和相关机理。在以上内容的基础上,我们发现,飞秒激光可以进一步调节细胞内的多种分子行为。通过精细地调节飞秒激光曝光刺激,可使其对He La细胞内钙离子进行释放或维持细胞的高钙水平,这种机制可以使得钙调磷酸酶充分将转录因子NFAT去磷酸化,从而激活NFAT的表达链路,使其在光刺激之后入核,启动相应的下游基因的表达。我们在实验上实现了飞秒激光对转录因子NFAT入核过程的调控,作为原理性的验证,实验中使用了荧光素酶报告基因做了检测。进一步地,我们使用飞秒激光对细胞的刺激,可以直接调控细胞自身的TNF-α等基因的表达。更进一步地,我们将这种技术应用于人类间充质干细胞(MSCs)中,实验上实现了飞秒激光对间充质干细胞内钙离子浓度的调控,并且对干细胞内的转录因子Runx2、Osterix等具有一定的影响,这种光刺激方法在诱导干细胞分化领域具有一定的应用价值。然而,飞秒激光刺激对于细胞的损伤是不可忽略的。我们发现不管是飞秒激光的单次短时间刺激,还是低功率连续激光扫描,都会影响细胞自噬水平。我们系统性地观测了不同的激光刺激对于细胞自噬的影响,分析了两种刺激机制,论证了细胞自噬对于光刺激的敏感性。我们认为,激光对于细胞的刺激,即使相对安全,不会对细胞活性造成较大影响,但依然会引起细胞自噬等过程。在飞秒激光的刺激下,线粒体可能是自噬体膜的来源之一。由于飞秒激光作用于细胞属于超快行为,所以对超快行为的观测具有着重要的意义。本文最后介绍了STEAM、STAMP、FIRE等几种超快成像技术,论述了这些技术的技术特点、发展现状以及应用场合等问题。实验上参与设计搭建了新型FIRE成像系统,得到了荧光成像图样,并对实验中遇到的一些细节问题做了实验上的探索以及总结。本文的一系列工作探索了飞秒激光对细胞刺激带来的影响,为光学方法对细胞过程和功能的调控提供了全新的理论和技术基础。
[Abstract]:Biophotonics is the leading field of optical and biological high-crossing, and the progress of femtosecond laser technology provides a series of new technologies, new methods, new ideas and new theories for biophotonics. At present, the biological photonics based on femtosecond laser have been deeply studied and widely used in many fields such as multi-photon imaging, cell micro-operation and so on. However, the effects of the femtosecond laser stimulation on the cell and the mechanism of the damage are relatively rough, the mechanism of the femtosecond laser pulse and the cell interaction, and the stress response of the cell to the femtosecond laser stimulation have not been clearly set forth. Under such a background, the influence of femtosecond laser stimulation on the cell process and the regulation mechanism were studied. The new method of femtosecond laser to regulate the intracell molecular behavior was put forward, and the ultra-fast imaging technique was further developed. In this paper, the progress of the bio-photonics based on femtosecond laser is reviewed, and the key problems and the development status of the research are reviewed in recent years. Then, as the basic theory of the related biological analysis, the related background of the calcium ion is briefly introduced, including the storage of calcium ion, the physiological function, the related molecular signal and so on. In this paper, a micro-operating system based on femtosecond laser, confocal microscope and CCD is introduced, and the experimental phenomena and related mechanism of the change of calcium ion concentration in the cell using femtosecond laser are discussed. On the basis of the above, we have found that the femtosecond laser can further regulate the various molecular behaviors in the cell. by finely adjusting the femtosecond laser exposure stimulus, it can release or maintain the high calcium level of the cells on the calcium ions in the He La cells, allowing it to enter the nucleus after the light stimulus to initiate the expression of the corresponding downstream gene. In the experiment, the regulation of the nuclear process of the transcription factor NFAT by the femtosecond laser is realized, and the luciferase reporter gene is used as the detection in the experiment. Further, we use the femtosecond laser to stimulate the cell, which can directly control the expression of the TNF-1 gene of the cell itself. Further, we apply this technique to the human mesenchymal stem cells (MSCs), and the effect of the femtosecond laser on the calcium ion concentration in the mesenchymal stem cells is realized, and the transcription factor Runx2, Osterx and the like in the stem cells have a certain influence, The photostimulation method has certain application value in the field of inducing stem cell differentiation. However, femtosecond laser stimulation is not negligible for cell damage. We found that both a single short-time stimulus of a femtosecond laser or a low-power continuous laser scan would affect the level of autophagy. We systematically observed the effects of different laser stimuli on the autophagy of the cells, and analyzed the two stimulation mechanisms, and demonstrated the sensitivity of the autophagy to the photostimulation. In our opinion, the stimulation of the laser to the cells, even relatively safe, does not have a great effect on the cell activity, but it still causes the cell autophagy and the like. Under the stimulation of femtosecond laser, the mitochondria may be one of the source of autophagy. It is of great significance to observe the ultra-fast behavior because the femtosecond laser is applied to the super-fast behavior of the cell. In this paper, several super fast imaging technologies, such as STEAM, STAMP, FIRE, etc., are introduced in this paper. The technical features, development status and application of these technologies are discussed. In the experiment, a new type of FIRE imaging system was built, and the fluorescence imaging pattern was obtained, and some of the detailed problems encountered in the experiment were explored and summarized. A series of work in this paper explores the influence of femtosecond laser on the cell stimulation, and provides a new theoretical and technical basis for the regulation of the process and function of the cell by the optical method.
【学位授予单位】：天津大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q27;TN249

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