细胞膜表面糖识别芯片制备及其初步应用的研究

发布时间：2018-10-11 06:37

【摘要】： 目的 糖、蛋白质、核酸是涉及生命活动本质的三类重要生物分子。随着分子生物学及细胞生物学的发展,糖的诸多生物功能不断被认识。糖可以以多糖或游离寡糖以及糖复合物的形式参与许多重要的生命活动。此外,还与癌症、细菌和病毒感染等疾病有着密切的关系,有关糖的研究将成为未来生命科学的中心研究课题之一。 目前,糖生物学研究中要解决的关键技术问题是糖链结构分析和合成方法的建立。为此,各国科学家都致力于糖链结构分析和合成方法学的研究,为糖链功能分析提供技术支持,同时糖链功能研究又集中在细胞水平上糖链的调控和识别机制的研究。 凝集素是一类能与糖专一非共价可逆结合的蛋白,凝集素的最大特点在于它们能识别糖及其复合物中复杂的碳水化合物结构,由于凝集素具有高度的糖结合专一性,它已经成为生物科学和临床科学非常有用的研究工具。目前利用凝集素进行细胞中糖链检测的方法有很多,如凝集素沉淀法、凝集素亲和层析法、凝集素亲和电泳法、凝集素酶联免疫吸附实验、凝集素印记法等,上述方法存在一个共同的缺点:只能对细胞中提取的糖蛋白进行测定,不能直接对细胞膜上的糖链进行分析。国外学者用凝集素芯片只对糖蛋白进行检测,没有进行细胞的捕获,需要对糖蛋白进行提纯,步骤繁琐,成本昂贵,且不能对细胞膜表面的糖蛋白进行直接的检测。 本研究旨在利用芯片的高通量性,制备一种能够检测糖蛋白上的糖链和不同组织来源细胞膜表面糖链特异性的凝集素芯片。由于该芯片能直接进行细胞捕获,不需要对细胞膜中的糖蛋白进行提取,成本低,操作简便快捷。通过检测芯片上凝集素各点阵所捕获细胞的荧光强度可以对糖蛋白上的糖链进行检测并对细胞膜表面糖链的分布获得总体的认识,因此,凝集素芯片的研制为观察和检测细胞膜表面糖链的种类及结构等研究建立了一个新的技术平台。 方法 1、凝集素芯片的制备 分别从芯片载片的不同修饰条件、凝集素在芯片上的固定效果检验、凝集素芯片捕获细胞能力几个方面进行研究。确定芯片载片的最佳修饰条件、最佳点样浓度。 2、凝集素芯片的初步应用 对凝集素芯片检测不同组织来源的细胞膜糖链特异性进行研究,确定凝集素芯片的应用价值。 3、凝集素芯片的检测 分别对凝集素芯片的稳定性、敏感性进行检测。 结果 1、凝集素芯片的制备 实验结果表明,凝集素芯片的最佳修饰条件是:硅烷溶液pH=7.2,水化时间20分钟,芯片载片经醛基修饰,凝集素点样浓度为1.4mg/ml。 2、凝集素芯片的初步应用 实验结果表明,凝集素芯片可以用于检测不同组织来源的细胞膜糖链特异性。 3、凝集素芯片的检测 实验结果表明,按照上述方法制备的芯片稳定性好,敏感度检测极限为1.8×10~6个/ml。 结论 本研究制备了一种能够检测糖蛋白上的糖链和不同组织来源细胞膜表面糖链特异性的凝集素芯片。该芯片稳定,重现性高,既具备芯片的高通量性,又避开碳水化合物芯片所面临的糖立体结构的复杂性。同时,还能直接进行细胞捕获,不需要对细胞膜中的糖蛋白进行提取,成本低,操作简便快捷。这为进一步研究细胞膜表面糖链结构、分析细胞免疫、细胞信号传递及进行糖组研究建立一个新的技术平台。也为应用凝集素芯片检测肿瘤细胞膜糖链的研究奠定了坚实的基础。
[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，

本文编号：2263212