【摘要】： 目的 糖、蛋白質(zhì)、核酸是涉及生命活動本質(zhì)的三類重要生物分子。隨著分子生物學(xué)及細(xì)胞生物學(xué)的發(fā)展,糖的諸多生物功能不斷被認(rèn)識。糖可以以多糖或游離寡糖以及糖復(fù)合物的形式參與許多重要的生命活動。此外,還與癌癥、細(xì)菌和病毒感染等疾病有著密切的關(guān)系,有關(guān)糖的研究將成為未來生命科學(xué)的中心研究課題之一。 目前,糖生物學(xué)研究中要解決的關(guān)鍵技術(shù)問題是糖鏈結(jié)構(gòu)分析和合成方法的建立。為此,各國科學(xué)家都致力于糖鏈結(jié)構(gòu)分析和合成方法學(xué)的研究,為糖鏈功能分析提供技術(shù)支持,同時糖鏈功能研究又集中在細(xì)胞水平上糖鏈的調(diào)控和識別機(jī)制的研究。 凝集素是一類能與糖專一非共價可逆結(jié)合的蛋白,凝集素的最大特點(diǎn)在于它們能識別糖及其復(fù)合物中復(fù)雜的碳水化合物結(jié)構(gòu),由于凝集素具有高度的糖結(jié)合專一性,它已經(jīng)成為生物科學(xué)和臨床科學(xué)非常有用的研究工具。目前利用凝集素進(jìn)行細(xì)胞中糖鏈檢測的方法有很多,如凝集素沉淀法、凝集素親和層析法、凝集素親和電泳法、凝集素酶聯(lián)免疫吸附實(shí)驗(yàn)、凝集素印記法等,上述方法存在一個共同的缺點(diǎn):只能對細(xì)胞中提取的糖蛋白進(jìn)行測定,不能直接對細(xì)胞膜上的糖鏈進(jìn)行分析。國外學(xué)者用凝集素芯片只對糖蛋白進(jìn)行檢測,沒有進(jìn)行細(xì)胞的捕獲,需要對糖蛋白進(jìn)行提純,步驟繁瑣,成本昂貴,且不能對細(xì)胞膜表面的糖蛋白進(jìn)行直接的檢測。 本研究旨在利用芯片的高通量性,制備一種能夠檢測糖蛋白上的糖鏈和不同組織來源細(xì)胞膜表面糖鏈特異性的凝集素芯片。由于該芯片能直接進(jìn)行細(xì)胞捕獲,不需要對細(xì)胞膜中的糖蛋白進(jìn)行提取,成本低,操作簡便快捷。通過檢測芯片上凝集素各點(diǎn)陣所捕獲細(xì)胞的熒光強(qiáng)度可以對糖蛋白上的糖鏈進(jìn)行檢測并對細(xì)胞膜表面糖鏈的分布獲得總體的認(rèn)識,因此,凝集素芯片的研制為觀察和檢測細(xì)胞膜表面糖鏈的種類及結(jié)構(gòu)等研究建立了一個新的技術(shù)平臺。 方法 1、凝集素芯片的制備 分別從芯片載片的不同修飾條件、凝集素在芯片上的固定效果檢驗(yàn)、凝集素芯片捕獲細(xì)胞能力幾個方面進(jìn)行研究。確定芯片載片的最佳修飾條件、最佳點(diǎn)樣濃度。 2、凝集素芯片的初步應(yīng)用 對凝集素芯片檢測不同組織來源的細(xì)胞膜糖鏈特異性進(jìn)行研究,確定凝集素芯片的應(yīng)用價值。 3、凝集素芯片的檢測 分別對凝集素芯片的穩(wěn)定性、敏感性進(jìn)行檢測。 結(jié)果 1、凝集素芯片的制備 實(shí)驗(yàn)結(jié)果表明,凝集素芯片的最佳修飾條件是:硅烷溶液pH=7.2,水化時間20分鐘,芯片載片經(jīng)醛基修飾,凝集素點(diǎn)樣濃度為1.4mg/ml。 2、凝集素芯片的初步應(yīng)用 實(shí)驗(yàn)結(jié)果表明,凝集素芯片可以用于檢測不同組織來源的細(xì)胞膜糖鏈特異性。 3、凝集素芯片的檢測 實(shí)驗(yàn)結(jié)果表明,按照上述方法制備的芯片穩(wěn)定性好,敏感度檢測極限為1.8×10~6個/ml。 結(jié)論 本研究制備了一種能夠檢測糖蛋白上的糖鏈和不同組織來源細(xì)胞膜表面糖鏈特異性的凝集素芯片。該芯片穩(wěn)定,重現(xiàn)性高,既具備芯片的高通量性,又避開碳水化合物芯片所面臨的糖立體結(jié)構(gòu)的復(fù)雜性。同時,還能直接進(jìn)行細(xì)胞捕獲,不需要對細(xì)胞膜中的糖蛋白進(jìn)行提取,成本低,操作簡便快捷。這為進(jìn)一步研究細(xì)胞膜表面糖鏈結(jié)構(gòu)、分析細(xì)胞免疫、細(xì)胞信號傳遞及進(jìn)行糖組研究建立一個新的技術(shù)平臺。也為應(yīng)用凝集素芯片檢測腫瘤細(xì)胞膜糖鏈的研究奠定了堅(jiān)實(shí)的基礎(chǔ)。
[Abstract]:Purpose Sugar, protein and nucleic acid are three kinds of important students involved in the essence of life activities With the development of molecular biology and cell biology, many biological functions of sugar continue It is recognized that sugars can be involved in many important plants in the form of polysaccharides or free oligosaccharides as well as sugar complexes In addition, it is closely related to diseases such as cancer, bacteria and viral infections, and research on sugar will become the center of the life sciences in the future At present, the key technology problem to be solved in the research of sugar chain is the analysis of sugar chain structure. To this end, scientists have devoted themselves to the research of sugar chain structure analysis and synthesis methodology, provide technical support for the functional analysis of sugar chain, and also concentrate on the regulation of sugar chain in cell level. A study of the mechanism of recognition. Lectin is a class of proteins that can bind to non-covalently reversibly bound to sugar. The largest feature of the lectin is that they can identify complex carbohydrate structures in sugars and their complexes, as a result of lectin Having a high degree of sugar binding specificity, it has become an essential factor and clinical significance. There are many methods for the detection of sugar chain in cells using lectins, such as lectin precipitation, lectin affinity chromatography, lectin affinity electrophoresis, lectin enzyme-linked immunosorbent assay, lectin imprinting, and the like. The method has a common disadvantage: only the glycoprotein extracted from the cells can be measured, The sugar chain on the cell membrane is analyzed. The foreign scholar uses the lectin chip only to detect the glycoprotein, does not carry out the cell capture, needs to purify the glycoprotein, the procedure is complicated, the cost is expensive, The purpose of this study is to make use of the high throughput of the chip, to prepare a kind of sugar chain that can detect the glycoprotein and the source of different tissues. The cell membrane surface sugar chain specific lectin chip. Because the chip can directly carry out cell capture, the glycoprotein in the cell membrane is not needed by detecting the fluorescence intensity of the cells captured by the lectin on the chip, the sugar chain on the glycoprotein can be detected and the surface of the cell membrane can be detected, The distribution of sugar chain is generally recognized. Therefore, the development of lectin chip is to observe and detect the type and knot of sugar chain on the surface of cell membrane. structure and so on The research set up a new technique The preparation method comprises the following steps: 1, preparing a lectin chip from different modification conditions of a chip carrier sheet, The results showed that the lectin chip was able to capture the cell capacity. The study was carried out to determine the chip load best modification conditions for tablets, optimal spotting concentration. 2, preliminary application of lectin chips to lectin cores To detect different tissue sources. Studies on the specificity of cell membrane sugar chain and the determination of lectin chips Application Value. 3, setting The stability and sensitivity of the lectin chip were tested, and the results showed that the lectin chip the best modification conditions are: silanolysis pH of liquid = 7.2, hydration time 20 min, chip carrier modified by aldehyde group, lectin spotting concentration 涓，

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