多种金属离子的单细胞分析及细胞膜蛋白的单分子检测

发布时间：2018-05-31 14:30

  本文选题：单细胞 + 金属离子　； 参考：《山东师范大学》2017年硕士论文

【摘要】：在生物体内,各种各样的生物分子发挥着至关重要的作用。如基因能够调控信号传递及代谢通路,蛋白质组学则在一定程度上反应基因表达水平,金属离子在神经系统中能够促进神经递质的释放和分泌、调节神经突触的形成及生长等。各种生物分子之间彼此关联、协同作用、相互影响,一种生物分子的异常表达往往会引起一系列的变化,因此各种生物分子与细胞功能的正常运作、各种疾病的发生与发展以及相关治疗密不可分。目前已发展了多种针对细胞内生物分子的研究方法,包括电化学检测、质谱以及光学检测等,其中荧光法由于检测灵敏度高等优点已得到广泛应用。但是目前的方法对于单细胞水平甚至是单分子水平的生物分子的检测仍然存在许多困难,如生物分子的尺度一般为纳米级,检测方法需具有较高的空间分辨;某些分子含量较少,低灵敏度的方法难以实现其精确的检测;另外由于细胞成分较为复杂,如何消除其他分子的信号干扰也是一大挑战。因此,急需发展高灵敏的单细胞内生物分子检测方法。针对这些困难与挑战,本论文致力于发展高灵敏度的检测方法用于细胞内生物分子的检测。首先我们在单细胞的水平上,发展了一种单细胞内多组分的同时定量检测的方法,并且进一步提高了检测灵敏度,构建了一种单分子水平上的细胞膜蛋白的检测方法。论文主要包括以下四个部分:第一章为绪论,首先概述了目前细胞内各种生物分子检测的研究进展,其中包括生物分子的重要生理功能及荧光检测方法的简介;随后介绍了单细胞水平上的生物分子的检测重要性及单细胞内多种金属离子同时定量检测的重要性,并总结了用于单细胞检测的微流控方法的优势;最后介绍了单分子检测的研究进展以及单分子水平上细胞膜蛋白检测的意义及方法。第二章介绍了基于目前单细胞水平上多种金属离子检测的困难,为了解决这些困难我们发展了一种基于微流控系统同时定量检测单个神经细胞内Na~+,K~+,Ca~(2+),Mg~(2+)四种金属离子的新方法,并将该方法应用于研究Aβ25-35诱导的PC-12细胞内四种离子的变化情况。该方法为单细胞内多种金属离子的研究提供了一种新的思路,同时研究结果也为多种金属离子如何在神经疾病的调节中发挥协同作用提供了新的启示。第三章在单细胞检测的基础上,进一步提高检测灵敏度,发展了一种单分子水平上的细胞膜蛋白的检测方法。选取了细胞膜蛋白PTK7作为研究对象,利用核酸适配体高效靶向目标膜蛋白,并通过引入滚环扩增反应来实现信号的放大,从而发展一种灵敏度好、毒性小、可进行细胞原位单分子成像检测的方法。该方法的检测灵敏度也能够满足低丰度的细胞膜蛋白的单分子检测。最后对本论文所建立的单细胞及单分子检测方法进行了总结。本论文在提高检测灵敏度的角度上,针对单个细胞内的多种金属离子实现了同时定量检测,并进一步提高检测灵敏度,研究了单分子水平上的细胞膜蛋白。这两种方法逐渐递进,本质都是针对细胞内生物分子的研究。而基于本论文建立的方法,细胞内其他生物分子的单细胞及单分子水平的检测还需更进一步的探索。
[Abstract]:In organisms, a variety of biomolecules play a vital role. For example, genes can regulate signaling and metabolic pathways, and proteomics reacts to the level of gene expression to a certain extent. Metal ions can promote the release and secretion of neurotransmitters in the nervous system, regulate the formation and growth of synapses. The abnormal expression of a biological molecule often causes a series of changes, so the normal operation of biological molecules and cell function, the occurrence and development of various diseases and related treatments are inseparable. The methods of research, including electrochemical detection, mass spectrometry and optical detection, have been widely used because of the high sensitivity of detection. However, there are still many difficulties in the detection of single cell level and even the single molecule level of biomolecules, such as the scale of biomolecules is generally nanoscale. It is necessary to develop a high sensitive method for detecting the signal interference of other molecules. Therefore, it is urgent to develop a highly sensitive single cell biomolecular detection method. With the challenge, this thesis is devoted to the development of highly sensitive detection methods for detection of intracellular biomolecules. First, we developed a simultaneous quantitative detection method for single cell multiple components at the single cell level, and further improved detection sensitivity, and constructed a single molecule level of cell membrane protein detection. This paper mainly includes the following four parts: the first chapter is the introduction. First, the research progress of various biological molecules detection in cells, including the important physiological functions of biomolecules and the introduction of fluorescence detection methods, is introduced. Then the importance of detection of biomolecules on single cell level and a variety of single cells are introduced. The importance of simultaneous quantitative detection of metal ions and the advantages of microfluidic methods used in single cell detection are summarized. Finally, the research progress of single molecule detection and the significance and methods of cell membrane protein detection on single molecule level are introduced. The second chapter introduces the difficulties of detecting various metal ions on the single cell level at present. To solve these difficulties, we developed a new method for the simultaneous quantitative detection of four metal ions, Na~+, K~+, Ca~ (2+), Mg~ (2+) in single neural cells, based on the microfluidic system, and applied this method to study the changes of four ions in PC-12 cells induced by A beta 25-35. This method is the study of a variety of metal ions in a single cell. A new idea is provided, and the results also provide new inspiration for how many metal ions play synergistic role in the regulation of neural diseases. In the third chapter, on the basis of single cell detection, the detection method of cell membrane protein on a single molecule level is developed. Protein PTK7 is used as a research object. The target membrane protein is highly targeted by the aptamer of nucleic acid, and the signal amplification is realized by introducing the rolling ring amplification reaction to develop a sensitive, small toxic method for the detection of cell in situ single molecule imaging. The detection sensitivity of this method can also satisfy the low abundance of cell membrane protein. Single molecule detection. Finally, the single cell and single molecule detection methods established in this paper are summarized. In this paper, the simultaneous quantitative detection of a variety of metal ions in a single cell is carried out at the angle of improving the detection sensitivity, and the detection sensitivity is further improved, and the cell membrane protein on the single molecule level is studied. The two methods are progressively progressive, and the essence is to study the intracellular biomolecules. Based on the method established in this paper, the detection of single cell and single molecular level of other biomolecules in the cell needs to be further explored.
【学位授予单位】：山东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q2-33;O657.3
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本文编号：1960094