DNA纳米技术在细胞动态控制和细胞动态成像中的应用

发布时间：2018-02-01 17:01

本文关键词： 细胞黏附基底细胞释放逻辑门基因位点多色成像 CRISPR-dcas9端粒 STED　出处：《中国科学院研究生院(上海应用物理研究所)》2016年博士论文　论文类型：学位论文

【摘要】：细胞外基质除了为细胞提供机械支撑之外,在多种生理病理过程中都具有重要作用。目前为止人们已经开发出各种各样的基底用于研究细胞的黏附以及细胞外基质组分的功能。然而在众多的方法中,精确控制基质中相关配体的密度和取向仍然是一个巨大的挑战。在本文的研究中,我们将金表面DNA自组装单层发展成了一种可以便捷的控制细胞黏附,实现细胞图案化的基底。在研究中我们发现金表面DNA自组装单层中由组装特性不同所引起的DNA链取向的差异影响着细胞的黏附行为。而且在研究中我们也证实了DNA自组装单层用做细胞培养基底的潜在可能。另外,通过改变DNA序列中poly A的长度,我们可以控制自组装单层中DNA密度来控制细胞黏附。使用DNA核酸适配体所构建的自组装单层,界面可以感应外界环境的变化调控细胞黏附。结合DNA点样仪,我们还实现了细胞的图案化,构建出细胞阵列以及字母形细胞图案。我们的结果表明金表面DNA自组装单层具有开发成智能界面的潜在可能,可以用在细胞以及组织工程的相关研究中。控制细胞释放是细胞培养中的重要步骤,同时在体内很多生理病理过程中都涉及到细胞释放的过程,因此控制细胞释放在生物学以及生物医学等领域的研究中具有重要的意义。在体内,控制细胞释放的过程受到多种因素的控制,不同因素之间相互作用,最终引起细胞的释放。在本文中,我们在体外构建了一个细胞黏附的基底,同时我们证实DNA链置换反应可以用来实现细胞释放。该过程对细胞没有损伤,而且反应迅速。通过设计DNA序列,我们还构建了各种逻辑门来控制细胞释放以模拟体内不同因素之间的协同拮抗等作用。CRISPR-cas9系统是人们在细菌以及古细菌中发现的一种特殊的免疫机制。因为该体系的特异性和高效性,人们将该体系开发成了一种基因编辑的工具,并已经在多种生物体中成功实现了目的基因的编辑。之后通过将cas蛋白进行突变以及融合荧光蛋白,该体系又被应用到活细胞内基因位点的标记,实现了靶标基因的动态观察。在本文中,我们提出了一种基因位点多色标记的策略,通过对不同sgRNA进行体外荧光标记,我们可以对细胞内不同基因位点进行多色标记。加上我们标记方法对细胞没有损伤,我们还可以对活细胞内基因位点进行动态追踪。我们认为该方法是一个研究活细胞内基因空间分布与动态运动的有效手段,是研究基因动态调控的有效工具。自从被发现一来,端粒在细胞内的重要作用就引起了人们的广泛关注。目前荧光显微观察是研究端粒的主要方法。然而普通荧光显微镜的分辨率受到了光学衍射极限的限制。近年来超分辨显微术的发展使人们可以在更高的分辨率水平上对目标进行观察。在本文中,我们使用受激发射损耗显微镜对间期细胞核中荧光标记的端粒进行了观察。结果表明,与激光共聚焦荧光显微镜相比,使用受激发射损耗显微镜所得到的荧光图片中端粒荧光斑点的尺寸更加小。而且一些由于位置较近而无法区分开来的端粒在超分辨图像中可以被清楚的区分开。因此,通过使用超分辨显微术,我们可以得到更多之前无法得到的关于端粒的信息。
[Abstract]:The extracellular matrix in addition to provide mechanical support for cells, plays an important role in many physiological and pathological processes. So far, people have developed a variety of substrates for the study of cell adhesion and extracellular matrix components. However, in these methods, density and orientation of precise control of related ligands in the matrix still it is a great challenge. In this study, we will DNA self-assembled monolayer on gold surface into a convenient control of cell adhesion, realize basal cell patterning. In this study we found that DNA self-assembled monolayer on gold surface by the difference of DNA chain orientation assembly properties caused by the different influence a cell adhesion behavior. But in the research we have demonstrated that DNA self-assembled monolayer cell culture with potential substrate. In addition, by changing the DNA sequence in poly A The length, we can control the density of DNA self assembled monolayer to control cell adhesion. The self-assembled monolayer aptamer constructed using DNA nucleic acid, the interface can change the regulation of cell adhesion induced by external environment. Combined with the DNA machine, we also realize cell patterning, construct cell array and cell pattern. Our alphabet the results show that the gold surface DNA has developed into a potential intelligent interface self assembled monolayer can be used in the study of cell and tissue engineering. Control cells release is an important step in cell culture and in vivo, many physiological and pathological processes are involved in the process of cell release, therefore has important implications in the control of cell the release of research in biology and biomedical and other fields. In vivo, control cells release is controlled by a variety of factors, different factors between each other The role, eventually lead to cell release. In this paper, we construct a basal cell adhesion in vitro, we confirmed that DNA strand displacement reaction can be used to achieve cell release. The process without damage to the cells, and rapid response. Through the design of the DNA sequence, we also construct a variety of logic gates to control the release of cells cooperative antagonism between different factors to simulate the in vivo effects of the.CRISPR-cas9 system is the people in the bacteria and archaea found in a special immune mechanism. Because of the specificity and efficiency of the system, people will develop the system into a gene editing tool, and has been successfully implemented in a variety of organisms the purpose of gene editing. After the CAS mutation and fusion protein fluorescent protein markers, this system has been applied to live cells loci, achieve target gene Dynamic observation. In this paper, we propose a gene locus multicolor labeling strategies were studied by in vitro fluorescent markers on different sgRNA, we can in the cells of different gene loci were multicolor labeling. Together our labeling method without damage to the cells, we can also be used in live cell gene loci for dynamic tracking. We believe that this method is an effective tool to study living cells of genes within the spatial distribution and dynamic motion, is a useful tool for dynamic regulation of genes. It was discovered that the important role of telomere in cells has attracted widespread attention. At present, fluorescence microscopy is the main method to study the telomere. However ordinary the resolution of fluorescence microscopy by optical diffraction limit. In recent years the development of super resolution microscopy can make people into the target in the higher resolution level For observation. In this paper, we use the emission of fluorescence microscopy telomere loss in interphase nuclei markers were observed. The results showed that compared with the laser confocal fluorescence microscopy, fluorescence spots using telomere fluorescence images of the STED microscope and the size of the more small. And some due to the location near to distinguish telomeres can be separated clearly in super resolution image. Therefore, by using super resolution microscopy, we can get more information about the telomere before can not be found.

【学位授予单位】：中国科学院研究生院(上海应用物理研究所)
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q813

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