蛋白质O-GlcNAc糖基化高灵敏比色检测分析及生物学功能研究

发布时间：2018-05-20 08:04

本文选题：O-GlcNAc糖基化 + 信号放大　；参考：《西南大学》2017年硕士论文

【摘要】：O-连接-N-乙酰葡萄糖胺(O-linked-N-acetyglucosamine,O-GlcNAc)糖基化是一种与磷酸化相似的、动态的、可逆的蛋白质翻译后修饰,由O-连接N-乙酰葡萄糖胺转移酶(OGT)和O-连接N乙酰葡萄糖胺水解酶(OGA)协同完成。OGT负责将GlcNAc基团添加到蛋白质的丝氨酸和苏氨酸残基上,OGA则将GlcNAc从蛋白质上移除。O-GlcNAc修饰和传统的糖基化不同,它主要发生在细胞核和细胞质中的蛋白质上,其糖链结构仅有一个氧糖苷键连接的N-乙酰葡萄糖胺单糖。在细胞内O-GlcNAc修饰和磷酸化直接或间接地相互作用,参与细胞的蛋白酶解、转录调控、信号转导、细胞周期调控等重要生命活动。近年来研究也表明,O-GlcNAc糖基化的异常与肿瘤、神经退行性疾病、心血管疾病、糖尿病等多种疾病密切相关。研究O-GlcNAc糖基化修饰在生命科学和生物医学中都具有重要意义。尽管研究者们开发了多种分析方法和检测手段,但是由于O-GlcNAc修饰的低丰度和动态变化的特点,O-GlcNAc的检测和研究受到了很大限制。国内外研究者围绕O-GlcNAc修饰与肿瘤的关系也开展了一些探索性研究,然而现有的报道主要集中于探讨细胞内总的O-GlcNAc糖基化水平,其结果并不能反映特定蛋白的O-GlcNAc修饰在肿瘤发生中的作用。另外,蛋白质O-GlcNAc糖基化修饰在调节肿瘤细胞增殖、迁移等相关信号通路中的作用和分子机制也还有待深入研究。针对目前O-GlcNAc糖基化修饰研究中的不足和空白,本文开发了一种基于铜沉积信号放大的O-GlcNAc糖基化高灵敏比色检测分析技术,同时应用微阵列芯片技术研究了前列腺细胞中特定蛋白质的O-GlcNAc糖基化,并进一步探讨了OGA抑制剂对前列腺细胞增殖、迁移的影响。本论文的主要研究内容和结果如下:1.基于铜沉积信号放大的O-GlcNAc糖基化比色检测方法的构建及性能评价在该设计中,麦胚凝集素(WGA)用作O-GlcNAc的特异识别分子,进而通过生物素-链霉亲和素系统将胶体金颗粒(AuNP)引入到传感器的表面。在还原剂抗坏血酸和硫酸铜存在的条件下,胶体金颗粒催化铜离子还原为铜并沉积于其表面,沉积铜的量与分析物的浓度成比例。其中AuNP催化(代替昂贵而不稳定的生物酶)的铜沉积能够有效地放大检测信号,形成超高的检测灵敏度。在加入氯化铁(FeCl3)后,沉积的铜被三价铁离子(Fe3+)氧化成Cu2+,同时Fe3+被还原成亚铁离子(Fe2+)。随后,通过Fe2+和红菲绕啉之间的配位产生红色络合物,引起溶液颜色的变化。因此,蛋白质O-GlcNAc修饰的水平可以通过用肉眼直接进行定性评价,或通过测量红色溶液的吸收值来定量检测。本文进一步以N-乙酰葡萄糖胺修饰的牛血清白蛋白(GlcNAc-conjugated BSA)为模型蛋白,对所开发的比色检测方法进行了评价。实验结果表明,GlcNAc-conjugated BSA的检测范围为0.1pg m L-1至10ng mL-1,最低检测限为0.02 pg mL-1,实现了蛋白质O-GlcNAc糖基化快速、超灵敏、低成本的分析检测。2.应用所开发比色检测方法总体上评价O-GlcNAc修饰与肿瘤的关系为了进一步评估上述比色检测的实际应用的可能性,本实验研究了前列腺癌细胞系(PC-3)和正常前列腺细胞系(RWPE-1)的细胞裂解液中的总O-GlcNAc糖基化水平。结果表明:与正常细胞系RWPE-1相比,癌细胞系PC-3的总O-GlcNAc糖基化水平显著升高,这与文献报道的结果一致。3.应用蛋白质微阵列芯片总体上评价O-GlcNAc修饰与肿瘤的关系以及验证OGT和OGA在O-GlcNAc修饰中的作用蛋白质微阵列芯片的结果表明:癌细胞系PC-3中的O-GlcNAc糖基化修饰水平显著高于正常细胞系RWPE-1中的水平,同时经OGA抑制剂Thiamet G处理的PC-3、RWPE-1细胞的裂解液中O-GlcNAc修饰水平均高于未处理细胞系,验证了抑制OGA的活性能够提高细胞内总O-GlcNAc修饰水平。此外,与正常前列腺细胞RWPE-1相比,前列腺癌细胞PC-3中OGT表达水平升高,OGA的表达水平降低。这些结果也表明,O-GlcNAc或许是一个很好的肿瘤分子标志物,可能在前列腺癌的诊断和治疗中起着重要作用。4.构建抗体微阵列芯片并用于肿瘤相关蛋白质的表达和O-GlcNAc修饰的并行分析本实验应用抗体微阵列芯片研究了细胞裂解液中c-Myc、NF-κB、p53三种蛋白的表达及其O-GlcNAc修饰水平,其中涉及经Thiamet G处理的PC-3、RWPE-1细胞和未经Thiamet G处理的PC-3、RWPE-1细胞。结果表明:与未经处理的细胞相比,OGA抑制剂处理后的两种细胞中的c-Myc、NF-κB蛋白O-GlcNAc糖基化修饰水平升高,而p53蛋白的O-GlcNAc糖基化修饰水平却降低,这说明OGA抑制剂促进了c-Myc、NF-κB两种蛋白的O-GlcNAc糖基化,抑制了p53蛋白的O-GlcNAc糖基化。并且与RWPE-1细胞系相比,PC-3细胞中c-Myc和p53的O-GlcNAc修饰水平降低,而NF-κB的O-GlcNAc修饰水平升高。5.OGA抑制剂对前列腺癌细胞增殖、迁移的影响为进一步研究O-GlcNAc糖基化水平对细胞生命活动的影响,本文通过细胞计数、MTT实验、细胞划痕实验等对细胞的增殖和迁移进行了评价。结果展示,经OGA抑制剂处理的细胞,其O-GlcNAc糖基化总水平升高,同时细胞增殖能力增强,迁移情况却没有明显改变。综上所述,本文开发了一种基于胶体金颗粒催化铜沉积进行信号放大的高灵敏显色检测技术,实现了低丰度蛋白质O-GlcNAc糖基化的检测;同时,研究了前列腺癌细胞中特定蛋白质的O-GlcNAc糖基化,探讨了OGA抑制剂对前列腺细胞增殖、迁移的影响,丰富了肿瘤和蛋白质糖基化方面的基础研究,并为肿瘤的靶向治疗和药物开发提供了理论依据。
[Abstract]:O- connection -N- acetyl glucosamine (O-linked-N-acetyglucosamine, O-GlcNAc) glycosylation is a kind of similar, dynamic, reversible protein translational modification that is similar to phosphorylation. O- connects N- acetyl glucosamine transferase (OGT) and O- connected N acetyl glucosamine hydrolase (OGA) together with.OGT responsible for adding GlcNAc group to protein. On the serine and threonine residues, OGA removes the GlcNAc from the protein and is different from the traditional glycosylation. It occurs mainly in the protein in the nucleus and cytoplasm, and its sugar chain structure is only a N- acetyl glucosamine monosaccharide connected by an oxyglucoside bond. O-GlcNAc modification and phosphorylation in the cell are direct or indirect. Interaction, involved in cell proteolysis, transcription regulation, signal transduction, cell cycle regulation and other important life activities. In recent years, studies have also shown that the abnormality of O-GlcNAc glycosylation is closely related to many diseases such as tumors, neurodegenerative diseases, cardiovascular diseases, diabetes and other diseases. O-GlcNAc glycosylation modification is studied in life science and biological medicine. Although the researchers have developed a variety of analytical methods and detection methods, the detection and research of O-GlcNAc have been greatly restricted because of the low abundance and dynamic changes of O-GlcNAc modification. Researchers at home and abroad have also carried out some exploratory studies on the relationship between O-GlcNAc modification and tumor. Some reports mainly focus on the total O-GlcNAc glycosylation level in cells, and the results do not reflect the role of O-GlcNAc modification of specific proteins in the carcinogenesis. In addition, the role and molecular mechanisms of protein O-GlcNAc glycosylation in regulating tumor cell proliferation, migration and other related signaling pathways are still to be studied. In view of the shortcomings and blanks in the study of O-GlcNAc glycosylation, a high sensitive colorimetric assay for O-GlcNAc glycosylation based on copper deposition signal amplification is developed, and microarray technology is used to study the O-GlcNAc glycosylation of specific proteins in the prostate cells, and the OGA inhibitors are further explored. The main contents and results of this paper are as follows: 1. the construction and performance evaluation of O-GlcNAc based glycosylated colorimetric assay based on copper deposition signal amplification in this design, wheat germ agglutinin (WGA) as a specific identification of O-GlcNAc, and then through biotin streptavidin system to colloid Jin Keli (AuNP) is introduced to the surface of the sensor. Under the condition of the reductant ascorbate and copper sulfate, the colloidal gold particles catalyze the reduction of copper ions to copper and deposited on the surface, and the amount of copper deposits is proportional to the concentration of the analyte. The copper deposition, which is catalyzed by AuNP (instead of the expensive and unstable biological enzymes), can be effectively amplified. After adding iron chloride (FeCl3), the deposited copper was oxidized to Cu2+ by Fe3+, and Fe3+ was reduced to ferrous ion (Fe2+). Then, the red complex was produced by the coordination between Fe2+ and red phenanthroline, and the color of the solution was changed. Therefore, the level of the protein O-GlcNAc modification. The qualitative evaluation can be done directly by the naked eye, or by measuring the absorption value of the red solution. In this paper, the N- acetyl glucosamine modified bovine serum albumin (GlcNAc-conjugated BSA) was used as a model protein to evaluate the developed colorimetric detection method. The experimental results showed that GlcNAc-conjugated BSA was found. The detection range is 0.1pg m L-1 to 10NG mL-1, the minimum detection limit is 0.02 PG mL-1, and the protein O-GlcNAc glycosylation rapid, sensitive, and low cost analysis and detection of.2. application development colorimetric detection method generally evaluates the relationship between O-GlcNAc modification and tumor in order to further evaluate the practical application of the above colorimetric detection. The total O-GlcNAc glycosylation level in the cell lysates of the prostate cancer cell line (PC-3) and the normal prostate cell line (RWPE-1) was studied. The results showed that the total O-GlcNAc glycosylation level of the cancer cell line PC-3 was significantly higher than that of the normal cell line RWPE-1, which was consistent with the results of the report, which was consistent with the.3. application protein microarray chip. The relationship between O-GlcNAc modification and tumor and the results of the function of OGT and OGA in O-GlcNAc modification showed that the level of O-GlcNAc glycosylation modification in the cancer cell line PC-3 was significantly higher than that in the normal cell line RWPE-1, while the PC-3 of the OGA suppressant Thiamet G, the lysate of the RWPE-1 cells The level of O-GlcNAc modification was higher than that of the untreated cell lines. It was shown that inhibition of OGA activity could increase the level of total O-GlcNAc modification in cells. In addition, the expression level of OGT in prostate cancer cell PC-3 was higher than that of normal prostate cell RWPE-1, and the expression level of OGA decreased. These results also suggest that O-GlcNAc may be a good tumor. Molecular markers may play an important role in the diagnosis and treatment of prostate cancer.4. construction of an antibody microarray and the parallel analysis of the expression of tumor related proteins and O-GlcNAc modification. The expression of three proteins of c-Myc, NF- kappa B, p53 and their O-GlcNAc modification in the cell lysate are studied by an antibody microarray. Levels, involving PC-3, RWPE-1 cells and PC-3, RWPE-1 cells treated by Thiamet G, and PC-3, RWPE-1 cells treated without Thiamet G. The results showed that, compared with the untreated cells, c-Myc of the two cells treated by the OGA inhibitor, NF- kappa B protein, increased the level of glycosylation modification, but the level of glycosylation modification was reduced. The OGA inhibitor promoted the O-GlcNAc glycosylation of two proteins of c-Myc, NF- kappa B and inhibited the O-GlcNAc glycosylation of p53 protein. Compared with RWPE-1 cell lines, c-Myc and p53 O-GlcNAc modification levels in PC-3 cells were reduced, and the inhibitory effect of the inhibitor on the proliferation and migration of prostate cancer cells was increased. The effect of O-GlcNAc glycosylation level on cell life activity was studied. The proliferation and migration of cells were evaluated by cell count, MTT experiment, and cell scratch test. The results showed that the total level of O-GlcNAc glycosylation level was increased by OGA inhibitors, and the cell proliferation ability was enhanced, and the migration situation was not. In summary, a high sensitive color detection technique based on colloidal gold particles catalyzed copper deposition was developed to detect the glycosylation of low abundance protein O-GlcNAc. At the same time, the O-GlcNAc glycosylation of specific proteins in prostate cancer cells was studied, and the OGA inhibitor on prostate cells was discussed. The effect of proliferation and migration enriches the basic research on the glycation of tumor and protein, and provides a theoretical basis for targeted therapy and drug development.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.3

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