發(fā)布時(shí)間：2018-01-12 13:29

  本文關(guān)鍵詞：平分型N-糖鏈在乳腺癌細(xì)胞及臨床樣本中的分子調(diào)控及生物學(xué)功能研究　出處：《江南大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 乳腺癌 平分型GlcNAc結(jié)構(gòu) β1 4-N-乙酰氨基葡萄糖轉(zhuǎn)移酶Ⅲ 糖組學(xué) 糖蛋白質(zhì)組學(xué)

【摘要】：糖基化是非常重要的蛋白質(zhì)翻譯后修飾之一,在多種生物過程中扮演重要角色,包括蛋白質(zhì)的折疊、蛋白質(zhì)的降解及細(xì)胞間的相互作用等。平分型N-乙酰葡萄糖胺(GlcNAc)結(jié)構(gòu)是在β1,4-N-乙酰氨基葡萄糖轉(zhuǎn)移酶Ⅲ(N-acetylglucosaminyltransferase Ⅲ,Gn T-Ⅲ or MAGT3)的催化下,將GlcNAc以β1,4-糖苷鍵連接到核心五糖中的甘露糖殘基上形成的。蛋白質(zhì)的平分型GlcNAc糖基化修飾對(duì)細(xì)胞內(nèi)信號(hào)轉(zhuǎn)導(dǎo)、細(xì)胞黏附、細(xì)胞遷移等生物過程都有重要作用。包括癌癥在內(nèi)的多種疾病中都存在異常的平分型GlcNAc糖基化。平分型GlcNAc結(jié)構(gòu)及MGAT3與腫瘤的遷移及侵襲等過程密切相關(guān)。但在乳腺癌的發(fā)生發(fā)展過程中,平分型GlcNAc結(jié)構(gòu)的變化及其生物學(xué)功能的全面性研究還鮮有報(bào)道,因此研究平分型GlcNAc結(jié)構(gòu)與MGAT3有助于深入了解其在乳腺癌發(fā)生發(fā)展過程中的作用,為乳腺癌的生物標(biāo)志物的研究及乳腺癌的治療提供一定的理論基礎(chǔ)。為此,本課題以乳腺癌為研究對(duì)象,全局性的分析平分型GlcNAc結(jié)構(gòu)的含量及MGAT3的表達(dá)變化,并鑒定平分型GlcNAc糖基化修飾的糖蛋白質(zhì),研究平分型GlcNAc結(jié)構(gòu)的生物學(xué)功能。主要研究結(jié)果如下:(1)EMT過程中糖組學(xué)分析。本實(shí)驗(yàn)首先建立了轉(zhuǎn)化生長(zhǎng)因子(TGFβ)誘導(dǎo)小鼠乳腺上皮(normal mouse mammary gland epithelial,NMu MG)細(xì)胞發(fā)生上皮間質(zhì)轉(zhuǎn)化(Epithelial-mesenchymal transition,EMT)的細(xì)胞模型,結(jié)合質(zhì)譜、基因芯片及凝集素芯片等高通量技術(shù),對(duì)EMT過程中異常糖基化及糖相關(guān)基因進(jìn)行研究,并利用凝集素染色及熒光定量PCR等技術(shù)進(jìn)行實(shí)驗(yàn)結(jié)果的驗(yàn)證。實(shí)驗(yàn)結(jié)果發(fā)現(xiàn),在EMT過程中,高甘露糖型N-糖鏈結(jié)構(gòu)含量升高;平分型GlcNAc結(jié)構(gòu)含量降低;巖藻糖糖基化程度降低;N-糖相關(guān)基因表達(dá)發(fā)生顯著變化,其中,糖相關(guān)基因ALG9與MGAT3表達(dá)下調(diào),糖相關(guān)基因轉(zhuǎn)錄水平變化與N-糖鏈的變化相一致。(2)乳腺癌發(fā)生發(fā)展過程中平分型GlcNAc結(jié)構(gòu)含量及MGAT3表達(dá)的異常變化。以人源、鼠源乳腺細(xì)胞株及人乳腺癌臨床樣本為實(shí)驗(yàn)材料,對(duì)乳腺癌中異常糖基化進(jìn)行質(zhì)譜檢測(cè),發(fā)現(xiàn)在乳腺癌細(xì)胞及乳腺癌臨床樣本中平分型GlcNAc結(jié)構(gòu)含量顯著降低;對(duì)含有30對(duì)乳腺癌組織的乳腺癌組織芯片進(jìn)行PHA-E熒光染色,發(fā)現(xiàn)23對(duì)乳腺癌組織中平分型GlcNAc結(jié)構(gòu)含量顯著降低;利用ONCOMINE數(shù)據(jù)庫、Western blot及免疫熒光染色等技術(shù)檢測(cè)發(fā)現(xiàn)乳腺癌中MGAT3的轉(zhuǎn)錄水平及蛋白水平表達(dá)下調(diào)。利用在線數(shù)據(jù)庫Meth HC分析發(fā)現(xiàn)MGAT3啟動(dòng)子區(qū)高甲基化,用去甲基化藥物(地西他賓,decitabine)處理細(xì)胞可以大幅提高M(jìn)GAT3的表達(dá)。利用在線繪制生存曲線的工具分析MGAT3對(duì)乳腺癌預(yù)后的影響,發(fā)現(xiàn)MGAT3高表達(dá)的乳腺癌患者無復(fù)發(fā)生存率較MGAT3低表達(dá)患者高。(3)利用PHA-E凝集素富集乳腺細(xì)胞系中平分型GlcNAc糖基化修飾的糖蛋白質(zhì),對(duì)蛋白質(zhì)進(jìn)行鑒定及功能解析。對(duì)人源及鼠源乳腺細(xì)胞系(人乳腺上皮細(xì)胞MCF10A、人乳腺癌細(xì)胞MCF7、SKBR3及MDA-MB-231;小鼠乳腺上皮細(xì)胞NMu MG、小鼠乳腺癌細(xì)胞4T1)中平分型GlcNAc糖基化修飾的靶蛋白進(jìn)行鑒定及其進(jìn)行生物信息學(xué)的分析,發(fā)現(xiàn)平分型GlcNAc糖基化修飾的靶蛋白有整合素(integrin)及表皮生長(zhǎng)因子受體(EGFR)等,涉及到的信號(hào)通路有ERK、AKT等。(4)建立了一種凝集素輔助的N-糖鏈的分離、富集方法。利用凝集素將高峰度糖鏈從低峰度糖鏈中分離、富集出來,再對(duì)兩個(gè)組分分別進(jìn)行純化、檢測(cè),以提高糖鏈檢測(cè)的靈敏度及覆蓋率。應(yīng)用本方法對(duì)標(biāo)準(zhǔn)蛋白(雞卵清白蛋白)及復(fù)雜生物樣品(NMu MG細(xì)胞蛋白及人血清)中N-糖鏈進(jìn)行分離、檢測(cè)后發(fā)現(xiàn)此方法能夠利用等量的蛋白質(zhì)同時(shí)對(duì)低峰度糖鏈及高峰度糖鏈進(jìn)行高靈敏度與高覆蓋率的檢測(cè)。
[Abstract]:Glycosylation is an important post-translational modification of proteins, play an important role in a variety of biological processes, including protein folding, protein degradation and cell interaction. N- bisecting GlcNAc transferase (GlcNAc) structure is in beta 1,4-N- acetylglucosamine (N-acetylglucosaminyltransferase III, Gn T- or MAGT3 III) under the catalysis of GlcNAc to beta 1,4- glycosidic bond to form mannose residues in the core of the five sugar. Bisecting GlcNAc protein glycosylation modification on intracellular signal transduction, cell adhesion, cell migration and other biological processes have an important role. Bisecting GlcNAc glycosylation there are a variety of unusual diseases including cancer. Closely related to the migration and invasion process of bisecting GlcNAc structure and MGAT3 with tumor. But in the development and progression of breast cancer, flat type Gl Study on the comprehensive changes in structure and biological function of cNAc has not been reported, so the study of bisecting GlcNAc structure and MGAT3 contribute to a deeper understanding of the process in its role in breast cancer development, providing a theoretical basis for the treatment of breast cancer and breast cancer biomarkers. Therefore, this topic with breast cancer as the research object, the expression change of MGAT3 content and analysis of split type GlcNAc structure of overall, sugar and protein identification bisecting GlcNAc glycosylation, biological function of bisecting GlcNAc structure. The main research results are as follows: (1) EMT in proteomics analysis. The first experiment the establishment of the transforming growth factor (TGF) induced mouse mammary epithelial (normal mouse mammary gland epithelial, NMu MG) cells of epithelial mesenchymal transition (Epithelial-mesenchymal transition, EMT) cell model, Combined with mass spectrometry, gene chip and lectin microarray high-throughput technology, research on genes related to abnormal glucose and sugar based EMT process, and the experimental results were verified by lectin staining and fluorescence quantitative PCR technique. The experimental results showed that in the EMT process, high dew sugar N- sugar chain structure was increased; reduce the content structure of bisecting GlcNAc; fucose glycosylated sugar decreased; expression of N- related genes changed significantly, including sugar related gene ALG9 and MGAT3 expression, consistent with changes of sugar related gene transcription level and N- sugar chain. (2) the abnormal expression of GlcNAc and MGAT3 split type structure content development process the occurrence of breast cancer. The source of clinical samples of murine breast cell line and human breast cancer as the experimental materials for mass spectrometric detection of aberrant glycosylation in breast cancer found in breast cancer cells and breast cancer Pro The content of GlcNAc in the sample split type bed structure decreased significantly; PHA-E staining with 30 of breast cancer and breast cancer tissue microarray, found that 23 of the bisecting GlcNAc in breast cancer tissue structure was significantly lower; using ONCOMINE database, Western blot and immunofluorescence staining techniques detected the transcription and protein level of MGAT3 in breast cancer expression. Using the online database Meth HC analysis showed that MGAT3 promoter hypermethylation, with demethylation drugs (decitabine, decitabine) cells can significantly improve the expression of MGAT3. MGAT3 analysis tools to draw survival curves available online on the prognosis of breast cancer, found no recurrence the survival rate was lower than that of MGAT3 expression in patients with high expression of MGAT3 in patients with breast cancer. (3) by bisecting PHA-E lectin enriched breast cell lines GlcNAc glycosylated protein sugar Qualitative analysis, identification and function of protein. In human and mouse mammary cells (human breast epithelial cell MCF10A, human breast cancer cell line MCF7, SKBR3 and MDA-MB-231; mouse mammary epithelial cells NMu MG mouse breast cancer cell 4T1) target protein Zhongping type GlcNAc glycosylation of identification and analysis bioinformatics, found that the target protein bisecting GlcNAc glycosylation of integrin (integrin) and epidermal growth factor receptor (EGFR) signaling pathway, involving ERK, AKT et al. (4) a separation, a lectin assisted N- sugar chain by lectin concentration method. The peak of sugar chains separated from low kurtosis sugar chain is concentrated, then the two components were purified, detection, in order to improve the sensitivity of detection of sugar chain and coverage. The application of the method of standard protein (ovalbumin (NMu) and complex biological samples The N- sugar chain was separated from MG cell protein and human serum. After detection, it was found that this method could detect the high sensitivity and high coverage of the low kurtosis sugar chain and the peak sugar chain by using the same amount of protein.

【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.9

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本文編號(hào)：1414480