本文選題：單細胞 + 金屬離子��； 參考：《山東師范大學》2017年碩士論文

【摘要】：在生物體內(nèi),各種各樣的生物分子發(fā)揮著至關(guān)重要的作用。如基因能夠調(diào)控信號傳遞及代謝通路,蛋白質(zhì)組學則在一定程度上反應(yīng)基因表達水平,金屬離子在神經(jīng)系統(tǒng)中能夠促進神經(jīng)遞質(zhì)的釋放和分泌、調(diào)節(jié)神經(jīng)突觸的形成及生長等。各種生物分子之間彼此關(guān)聯(lián)、協(xié)同作用、相互影響,一種生物分子的異常表達往往會引起一系列的變化,因此各種生物分子與細胞功能的正常運作、各種疾病的發(fā)生與發(fā)展以及相關(guān)治療密不可分。目前已發(fā)展了多種針對細胞內(nèi)生物分子的研究方法,包括電化學檢測、質(zhì)譜以及光學檢測等,其中熒光法由于檢測靈敏度高等優(yōu)點已得到廣泛應(yīng)用。但是目前的方法對于單細胞水平甚至是單分子水平的生物分子的檢測仍然存在許多困難,如生物分子的尺度一般為納米級,檢測方法需具有較高的空間分辨;某些分子含量較少,低靈敏度的方法難以實現(xiàn)其精確的檢測;另外由于細胞成分較為復(fù)雜,如何消除其他分子的信號干擾也是一大挑戰(zhàn)。因此,急需發(fā)展高靈敏的單細胞內(nèi)生物分子檢測方法。針對這些困難與挑戰(zhàn),本論文致力于發(fā)展高靈敏度的檢測方法用于細胞內(nèi)生物分子的檢測。首先我們在單細胞的水平上,發(fā)展了一種單細胞內(nèi)多組分的同時定量檢測的方法,并且進一步提高了檢測靈敏度,構(gòu)建了一種單分子水平上的細胞膜蛋白的檢測方法。論文主要包括以下四個部分:第一章為緒論,首先概述了目前細胞內(nèi)各種生物分子檢測的研究進展,其中包括生物分子的重要生理功能及熒光檢測方法的簡介;隨后介紹了單細胞水平上的生物分子的檢測重要性及單細胞內(nèi)多種金屬離子同時定量檢測的重要性,并總結(jié)了用于單細胞檢測的微流控方法的優(yōu)勢;最后介紹了單分子檢測的研究進展以及單分子水平上細胞膜蛋白檢測的意義及方法。第二章介紹了基于目前單細胞水平上多種金屬離子檢測的困難,為了解決這些困難我們發(fā)展了一種基于微流控系統(tǒng)同時定量檢測單個神經(jīng)細胞內(nèi)Na~+,K~+,Ca~(2+),Mg~(2+)四種金屬離子的新方法,并將該方法應(yīng)用于研究Aβ25-35誘導(dǎo)的PC-12細胞內(nèi)四種離子的變化情況。該方法為單細胞內(nèi)多種金屬離子的研究提供了一種新的思路,同時研究結(jié)果也為多種金屬離子如何在神經(jīng)疾病的調(diào)節(jié)中發(fā)揮協(xié)同作用提供了新的啟示。第三章在單細胞檢測的基礎(chǔ)上,進一步提高檢測靈敏度,發(fā)展了一種單分子水平上的細胞膜蛋白的檢測方法。選取了細胞膜蛋白PTK7作為研究對象,利用核酸適配體高效靶向目標膜蛋白,并通過引入滾環(huán)擴增反應(yīng)來實現(xiàn)信號的放大,從而發(fā)展一種靈敏度好、毒性小、可進行細胞原位單分子成像檢測的方法。該方法的檢測靈敏度也能夠滿足低豐度的細胞膜蛋白的單分子檢測。最后對本論文所建立的單細胞及單分子檢測方法進行了總結(jié)。本論文在提高檢測靈敏度的角度上,針對單個細胞內(nèi)的多種金屬離子實現(xiàn)了同時定量檢測,并進一步提高檢測靈敏度,研究了單分子水平上的細胞膜蛋白。這兩種方法逐漸遞進,本質(zhì)都是針對細胞內(nèi)生物分子的研究。而基于本論文建立的方法,細胞內(nèi)其他生物分子的單細胞及單分子水平的檢測還需更進一步的探索。
[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.
【學位授予單位】：山東師范大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q2-33;O657.3
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本文編號：1960094