【作者】 刘侠；

【导师】 李钰；

【作者基本信息】 哈尔滨工业大学 ， 生物医学工程， 2013， 博士

【摘要】 肝细胞癌（HCC）是全球最常见的恶性肿瘤之一，其病情进展快、预后差，因早期诊断困难、治疗手段有限而死亡率高。因此，研究其发生发展的分子基础，寻找HCC早期诊断生物标志物和潜在治疗靶点，对HCC的诊断和治疗具有重要意义。糖蛋白糖链参与细胞识别、信号传导、免疫监视，维持着多细胞生物体各项有序性生命活动。糖链的异常改变通常与多种疾病包括肿瘤的发生发展密切相关。对恶性肿瘤特异性糖链结构与功能的研究不仅可以揭示糖链的生物学功能，还能为肿瘤生物标志物的筛选和新药靶点的开发奠定基础。DSA-FACE技术是本世纪初新兴的糖生物学检测技术，因其高通量、高灵敏度的特点，目前已在肿瘤血清生物标志物研究中得到足够认识，但利用该技术对非血清样本的研究则少有报道。本文从糖生物学角度、利用DSA-FACE技术对HCC特异性N-糖链结构及其发生异常改变的分子机理进行研究，以期为HCC的早期诊断、恶性演进和转移预测提供生物标志物，为HCC靶向治疗靶点选择及基于糖链结构改变的恶性转化机制研究奠定基础。在研究中，首先优化了血清糖蛋白N-糖组DSA-FACE检测技术，有效提高了其检测效率。在此基础上，建立了可用于体外培养细胞系及临床组织样本细胞表面N-糖组分析技术，通过分析膜蛋白不同制备方法（去污剂法和超速离心法），并引入石墨化炭黑（GC）树脂纯化技术、优化研究体系，使得该技术对于细胞表面N-糖组的检测更加灵敏、可靠，并具有良好的可重复性，从而拓展了DSA-FACE技术的应用范围，突破了以往只用于检测血清样本N-糖组的局限性。在优化N-糖链检测技术的基础上，本文分析了北方地区190例HCC患者血清样本，以110例乙肝病毒感染者及179例健康人血清样本作为对照。结果发现，由血清中三个发生异常变化的N-糖链NGA2F、NGA2FB和NA3联合获得的Log[(NGA2F+NGA2FB)/NA3]指标对于HCC的诊断准确性可达85%，诊断灵敏度和特异性分别可达到71.1%和86.0%，且该指标的诊断准确性不受患者性别、年龄、肿瘤病理分期、癌细胞分化程度等因素影响。深入分析本研究中40例AFP处于正常值范围（AFP <20ng/mL）的HCC患者血清，结果显示有23例血清中Log[(NGA2F+NGA2FB)/NA3]指标明显高于阈值（0.1045），证明该指标可有效降低AFP对于HCC的漏诊率。进一步利用Logistic回归分析法建立基于血清特异性糖链（NGA2F、NGA2FB、NA3）与AFP的联合诊断模型，ROC曲线分析表明，联合诊断模型对于HCC的诊断准确性可达到94.8%。该结果证实，以血清N-糖链生物标志物为基础的联合诊断模型可作为我国北方地区HCC特异性的普查生物标志物。利用本文建立的细胞表面N-糖组DSA-FACE技术完成88对临床HCC组织样本细胞表面N-糖组分析。结果表明，与癌旁对照组相比，峰L10（NA3FB）在HCC细胞表面显著性增加（p<0.0001），而峰L6（M8）在HCC细胞表面显著性降低（p<0.0001）。这两个N-糖链在不同性别、年龄及癌细胞分化程度的患者中均表现出显著性改变，并于HCC发病早期就具有明显改变的趋势。此外，通过与具有不同组织来源及性别差异的100对乳腺癌组织细胞表面N-糖组的比较，NA3FB在两种不同的肿瘤细胞表面均具有显著性增高趋势，而NA2和M8的差异表达则具有明显的组织特异性。进一步通过分析61例HCC患者的临床组织样本及相应的血清样本N-糖组，发现，NA3FB不仅在HCC细胞表面显著性增高（p<0.0001），而且在相应患者血清中的含量也显著增高（p<0.0001）。这表明， HCC驻留型及分泌型糖蛋白NA3FB糖链修饰异常增多可能与HCC的发病机制具有相关性。为探讨NA3FB显著增加的分子机理，本文对能够合成NA3FB的五种糖基转移酶（Fut8、GalT-1、GnT-V、GnT-IVa、GnT-IVb）在HCC临床组织中的表达进行了分析。结果表明，与相应癌旁组织相比，肿瘤组织中只有GnT-IVamRNA及蛋白的表达水平具有增高趋势。利用RNAi技术减低mgat4a表达48h后，检测HCC细胞系HepG2细胞表面NA3FB的表达水平。结果表明，与阴性对照相比，基因沉默组细胞表面NA3FB显著减少。这说明NA3FB的减少与糖基转移酶GnT-IVa的基因沉默直接相关。因此，该结果证实HCC细胞中GnT-IVa的异常高表达与细胞表面及血清中NA3FB异常增加密切相关。综上所述，本文通过分析膜蛋白制备方法及引入GC树脂纯化方法，建立了适合于不同临床样本N-糖链的DSA-FACE检测技术，获得了适用于中国北方地区HCC早期诊断的血清糖链生物标志物，发现了HCC细胞表面异常增加的糖链NA3FB与GnT-IVa的异常高表达密切相关。由此，这种糖基化异常机理的初步分析为深入研究糖基转移酶与糖链结构异常、与HCC发生发展的关系提供了新的思路，对HCC早期诊断和潜在靶向治疗的靶点选择奠定了基础。更多还原

【Abstract】 Hepatocellular carcinoma (HCC) is one of the most common cancers worldwideand has a high mortality rate due to the difficulty of early diagnosis and limitedtherapeutic methods. Therefore, it becomes critical to reveal the underlyingmolecular mechanisms of HCC development and to look for early diagnosticbiomarkers and treatment targets for HCC.The glycans of glycoproteins play pivotal roles in cell recognition, signaltransduction, immune surveillance and maintenance of ordered life activities ofmutilcellular organisms. Glycan alternations are closely associated with theinitiation and progression of many diseases including cancer. Research onstructures and functions of tumor-specific glycans will not only reveal thebiofunctions of glycans, but also enable the identification of new cancer biomarkersand drug targets. DSA-FACE (DNA sequencer-assisted fluorophore-assistedcarbohydrate electrophoresis) is an emerging glycobiological technique in thiscentury and has been widely used in the research of serum biomarkers for cancer.However, the application of this technique in non-serum specimens has rarely beenreported.This thesis studied the structure of HCC-specific N-glycans and associatedmolecular mechanisms using DSA-FACE technique. First of all, DSA-FACEtechnique used to detect N-glycans in serum was optimized and the detectivequantum efficiency has been remarkably promoted. Based on this, a DSA-FACEtechnique that can detect N-glycans in the in-vitro cultured cell lines and humanclinical tissues was developed. Compared to the conventional DSA-FACE, thisnewly developed technique has much higher stability, sensitivity and repeatability,which has been achieved by optimizing membrane protein extraction methods andintroducing GC resin purification procedure. This technique expands theapplication field of DSA-FACE and eliminates the limitation of conventionalDSA-FACE technique that only can be used for the glycan detection in serumsamples.Using the optimized serum DSA-FACE technique,190serum samples of HCCpatients were detected along with179serum samples of healthy humans and110serum samples of HBV-infected patients as controls. The results indicated thatLog[(NGA2F+NGA2FB)/NA3] which composed of3abnormal N-glycans, NGA2F,NGA2FB and NA3, in HCC patients are potential early diagnostic serumbiomarkers for HCC. The diagnostic efficiency of this biomarker is as high as85%with71.1%sensitivity and86.0%specificity. The diagnostic efficiency of this biomarker is independent of sex, age, tumor stage and differentiation degree ofHCC patients. Furthermore, the serum samples from23out of40HCC patientswhose AFP values are within normal range (AFP <20ng/ml) showed distinctlyhigher Log[(NGA2F+NGA2FB)/NA3] value above the threshod (0.1045), whichindicated that Log[(NGA2F+NGA2FB)/NA3] can effectively reduce the falsenegative rate of AFP detection. Thus, a HCC-specific glycans (NGA2F, NGA2FBand NA3)-AFP combined diagnosis model was further established using Logisticregression. The diagnostic efficiency of the combined diagnosis model for HCCreached94.8%as shown in ROC curve. This result supports the idea that the serumN-glycan biomarker-based combined diagnosis model can serve as a HCC-specificscreening biomarker in the north of P. R. China.Then, the cell surface N-glycan profiles of88pairs of HCC clinical tissues wereexamined by the newly established DSA-FACE technique which was suit for celllines and clinical tissues. The results indicated that the N-glycan peak L10(corresponding to NA3FB) was significantly increased (p<0.0001) on the cellsurface of HCC tissues compared to the paired adjacent normal tissues, while peakL6was significantly decreased (p<0.0001) on the cell surface of HCC tissues. Sametrend of alternations on both N-glycans were observed in all sex, age and celldifferentiation degree groups. In fact, these alterations have been found to presentat early stage of HCC. In addition, by comparison with the N-glycan profiles of100pairs breast cancer tissues we found that NA3FB was significant increased on thecell surface in a tissue and sex independent manner, while NA2and M8showeddistinct tissue specificity. The further comparative analysis of the clinical tissuesand corresponding serum samples in61HCC patients showed that NA3FB was notonly increased on the cell surface of HCC cells (p<0.0001), but also increased inthe corresponding serum samples (p<0.0001). This indicated that the abnormalincreased resident and excreted NA3FB glycoprotein was likely related to thetumorogenesis mechanisms of HCC.In order to investigate the molecular mechanisms of the significant enhancedNA3FB, both resident and excreted glycoproteins, in HCC patients, the expressionlevel of5glycosyltransferases (Fut8, GalT-1, GnT-V, GnT-IVa, GnT-IVb) whichcan produce NA3FB in HCC were analyzed. The results indicated that onlyGnT-Iva on both mRNA and protein levels were significantly increased in HCCtissues compared with paired adjacent tissues. The cell surface NA3FB in HepG2cells was detected48hours later after mgat4a (the gene of GnT-IVa) knockdown byusing RNAi technique. NA3FB was significantly decreased in response to mgat4aknockdown, suggesting that the decrease of NA3FB was related to the gene silence of GnT-IVa. Therefore, the enhanced GnT-IVa in HCC was closely related to theincreased levels of NA3FB both on HCC cell surface and in serum.To sum up, this study successfully established a N-glycan DSA-FACE techniquewhich was suit for the analysis of different clinical tissue specimens. The obtainedN-glycan serum biomarker can be used for the early diagnosis of HCC in the northarea of China. HCC-cell-surface-specific biomarker NA3FB was closely associatedwith the increased GnT-Iva in HCC. Therefore, the preliminary study on this alertedglycosylation mechanisms in HCC provides a new path for further research on therelationship of glycosyltransferases and glycan structure with the carcinogenesisand malignant progression of HCC. It also advanced the study on the earlydiagnosis and targeting therapy of HCC.更多还原