本文選題：O-GlcNAc糖基化 + 信號(hào)放大��； 參考：《西南大學(xué)》2017年碩士論文

【摘要】：O-連接-N-乙酰葡萄糖胺(O-linked-N-acetyglucosamine,O-GlcNAc)糖基化是一種與磷酸化相似的、動(dòng)態(tài)的、可逆的蛋白質(zhì)翻譯后修飾,由O-連接N-乙酰葡萄糖胺轉(zhuǎn)移酶(OGT)和O-連接N乙酰葡萄糖胺水解酶(OGA)協(xié)同完成。OGT負(fù)責(zé)將GlcNAc基團(tuán)添加到蛋白質(zhì)的絲氨酸和蘇氨酸殘基上,OGA則將GlcNAc從蛋白質(zhì)上移除。O-GlcNAc修飾和傳統(tǒng)的糖基化不同,它主要發(fā)生在細(xì)胞核和細(xì)胞質(zhì)中的蛋白質(zhì)上,其糖鏈結(jié)構(gòu)僅有一個(gè)氧糖苷鍵連接的N-乙酰葡萄糖胺單糖。在細(xì)胞內(nèi)O-GlcNAc修飾和磷酸化直接或間接地相互作用,參與細(xì)胞的蛋白酶解、轉(zhuǎn)錄調(diào)控、信號(hào)轉(zhuǎn)導(dǎo)、細(xì)胞周期調(diào)控等重要生命活動(dòng)。近年來(lái)研究也表明,O-GlcNAc糖基化的異常與腫瘤、神經(jīng)退行性疾病、心血管疾病、糖尿病等多種疾病密切相關(guān)。研究O-GlcNAc糖基化修飾在生命科學(xué)和生物醫(yī)學(xué)中都具有重要意義。盡管研究者們開(kāi)發(fā)了多種分析方法和檢測(cè)手段,但是由于O-GlcNAc修飾的低豐度和動(dòng)態(tài)變化的特點(diǎn),O-GlcNAc的檢測(cè)和研究受到了很大限制。國(guó)內(nèi)外研究者圍繞O-GlcNAc修飾與腫瘤的關(guān)系也開(kāi)展了一些探索性研究,然而現(xiàn)有的報(bào)道主要集中于探討細(xì)胞內(nèi)總的O-GlcNAc糖基化水平,其結(jié)果并不能反映特定蛋白的O-GlcNAc修飾在腫瘤發(fā)生中的作用。另外,蛋白質(zhì)O-GlcNAc糖基化修飾在調(diào)節(jié)腫瘤細(xì)胞增殖、遷移等相關(guān)信號(hào)通路中的作用和分子機(jī)制也還有待深入研究。針對(duì)目前O-GlcNAc糖基化修飾研究中的不足和空白,本文開(kāi)發(fā)了一種基于銅沉積信號(hào)放大的O-GlcNAc糖基化高靈敏比色檢測(cè)分析技術(shù),同時(shí)應(yīng)用微陣列芯片技術(shù)研究了前列腺細(xì)胞中特定蛋白質(zhì)的O-GlcNAc糖基化,并進(jìn)一步探討了OGA抑制劑對(duì)前列腺細(xì)胞增殖、遷移的影響。本論文的主要研究?jī)?nèi)容和結(jié)果如下:1.基于銅沉積信號(hào)放大的O-GlcNAc糖基化比色檢測(cè)方法的構(gòu)建及性能評(píng)價(jià)在該設(shè)計(jì)中,麥胚凝集素(WGA)用作O-GlcNAc的特異識(shí)別分子,進(jìn)而通過(guò)生物素-鏈霉親和素系統(tǒng)將膠體金顆粒(AuNP)引入到傳感器的表面。在還原劑抗壞血酸和硫酸銅存在的條件下,膠體金顆粒催化銅離子還原為銅并沉積于其表面,沉積銅的量與分析物的濃度成比例。其中AuNP催化(代替昂貴而不穩(wěn)定的生物酶)的銅沉積能夠有效地放大檢測(cè)信號(hào),形成超高的檢測(cè)靈敏度。在加入氯化鐵(FeCl3)后,沉積的銅被三價(jià)鐵離子(Fe3+)氧化成Cu2+,同時(shí)Fe3+被還原成亞鐵離子(Fe2+)。隨后,通過(guò)Fe2+和紅菲繞啉之間的配位產(chǎn)生紅色絡(luò)合物,引起溶液顏色的變化。因此,蛋白質(zhì)O-GlcNAc修飾的水平可以通過(guò)用肉眼直接進(jìn)行定性評(píng)價(jià),或通過(guò)測(cè)量紅色溶液的吸收值來(lái)定量檢測(cè)。本文進(jìn)一步以N-乙酰葡萄糖胺修飾的牛血清白蛋白(GlcNAc-conjugated BSA)為模型蛋白,對(duì)所開(kāi)發(fā)的比色檢測(cè)方法進(jìn)行了評(píng)價(jià)。實(shí)驗(yàn)結(jié)果表明,GlcNAc-conjugated BSA的檢測(cè)范圍為0.1pg m L-1至10ng mL-1,最低檢測(cè)限為0.02 pg mL-1,實(shí)現(xiàn)了蛋白質(zhì)O-GlcNAc糖基化快速、超靈敏、低成本的分析檢測(cè)。2.應(yīng)用所開(kāi)發(fā)比色檢測(cè)方法總體上評(píng)價(jià)O-GlcNAc修飾與腫瘤的關(guān)系為了進(jìn)一步評(píng)估上述比色檢測(cè)的實(shí)際應(yīng)用的可能性,本實(shí)驗(yàn)研究了前列腺癌細(xì)胞系(PC-3)和正常前列腺細(xì)胞系(RWPE-1)的細(xì)胞裂解液中的總O-GlcNAc糖基化水平。結(jié)果表明:與正常細(xì)胞系RWPE-1相比,癌細(xì)胞系PC-3的總O-GlcNAc糖基化水平顯著升高,這與文獻(xiàn)報(bào)道的結(jié)果一致。3.應(yīng)用蛋白質(zhì)微陣列芯片總體上評(píng)價(jià)O-GlcNAc修飾與腫瘤的關(guān)系以及驗(yàn)證OGT和OGA在O-GlcNAc修飾中的作用蛋白質(zhì)微陣列芯片的結(jié)果表明:癌細(xì)胞系PC-3中的O-GlcNAc糖基化修飾水平顯著高于正常細(xì)胞系RWPE-1中的水平,同時(shí)經(jīng)OGA抑制劑Thiamet G處理的PC-3、RWPE-1細(xì)胞的裂解液中O-GlcNAc修飾水平均高于未處理細(xì)胞系,驗(yàn)證了抑制OGA的活性能夠提高細(xì)胞內(nèi)總O-GlcNAc修飾水平。此外,與正常前列腺細(xì)胞RWPE-1相比,前列腺癌細(xì)胞PC-3中OGT表達(dá)水平升高,OGA的表達(dá)水平降低。這些結(jié)果也表明,O-GlcNAc或許是一個(gè)很好的腫瘤分子標(biāo)志物,可能在前列腺癌的診斷和治療中起著重要作用。4.構(gòu)建抗體微陣列芯片并用于腫瘤相關(guān)蛋白質(zhì)的表達(dá)和O-GlcNAc修飾的并行分析本實(shí)驗(yàn)應(yīng)用抗體微陣列芯片研究了細(xì)胞裂解液中c-Myc、NF-κB、p53三種蛋白的表達(dá)及其O-GlcNAc修飾水平,其中涉及經(jīng)Thiamet G處理的PC-3、RWPE-1細(xì)胞和未經(jīng)Thiamet G處理的PC-3、RWPE-1細(xì)胞。結(jié)果表明:與未經(jīng)處理的細(xì)胞相比,OGA抑制劑處理后的兩種細(xì)胞中的c-Myc、NF-κB蛋白O-GlcNAc糖基化修飾水平升高,而p53蛋白的O-GlcNAc糖基化修飾水平卻降低,這說(shuō)明OGA抑制劑促進(jìn)了c-Myc、NF-κB兩種蛋白的O-GlcNAc糖基化,抑制了p53蛋白的O-GlcNAc糖基化。并且與RWPE-1細(xì)胞系相比,PC-3細(xì)胞中c-Myc和p53的O-GlcNAc修飾水平降低,而NF-κB的O-GlcNAc修飾水平升高。5.OGA抑制劑對(duì)前列腺癌細(xì)胞增殖、遷移的影響為進(jìn)一步研究O-GlcNAc糖基化水平對(duì)細(xì)胞生命活動(dòng)的影響,本文通過(guò)細(xì)胞計(jì)數(shù)、MTT實(shí)驗(yàn)、細(xì)胞劃痕實(shí)驗(yàn)等對(duì)細(xì)胞的增殖和遷移進(jìn)行了評(píng)價(jià)。結(jié)果展示,經(jīng)OGA抑制劑處理的細(xì)胞,其O-GlcNAc糖基化總水平升高,同時(shí)細(xì)胞增殖能力增強(qiáng),遷移情況卻沒(méi)有明顯改變。綜上所述,本文開(kāi)發(fā)了一種基于膠體金顆粒催化銅沉積進(jìn)行信號(hào)放大的高靈敏顯色檢測(cè)技術(shù),實(shí)現(xiàn)了低豐度蛋白質(zhì)O-GlcNAc糖基化的檢測(cè);同時(shí),研究了前列腺癌細(xì)胞中特定蛋白質(zhì)的O-GlcNAc糖基化,探討了OGA抑制劑對(duì)前列腺細(xì)胞增殖、遷移的影響,豐富了腫瘤和蛋白質(zhì)糖基化方面的基礎(chǔ)研究,并為腫瘤的靶向治療和藥物開(kāi)發(fā)提供了理論依據(jù)。
[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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：O657.3

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本文編號(hào)：1913818