胃癌、肺癌特异性IgG Fc N-糖基化修饰标志物的研究

发布时间：2018-06-06 14:47

本文选题：疾病特异性IgG + Fc　；参考：《北京协和医学院》2017年博士论文

【摘要】：癌症是仅次于心血管疾病的人类健康第二大杀手,其中,胃癌和肺癌在癌症相关的发生率与死亡率中排名前列。癌症患者出现的早期症状如被误诊为相关炎症,则可能导致错过最佳的治疗时机而降低生存机会,所以良性疾病与癌症的诊断区分具有十分重要的临床意义。免疫球蛋白G(Immunoglobulin G,IgG)是人体内重要的免疫分子之一,其Fc N-糖基化修饰可以改变IgG分子与Fc受体之间的相互作用方式,从而影响IgG的促炎或抗炎活性,且与人类生理病理状态显著相关。然而前期研究主要关注血液总IgG,这在一定程度上忽视了抗原特异性IgG的结构变化。IgG作为外周血中丰度仅次于白蛋白的蛋白分子,并非所有IgG分子都与疾病发生、发展相关;前期研究结果表明,疾病发展过程中抗原特异性IgG糖基化修饰与血液总IgG糖基化修饰的变化趋势并不完全一致,所以研究抗原特异性IgG或疾病特异性IgG(Disease-specific IgG,DSIgG)能更精确反映病理状态。本研究采用非变性丙烯酰胺凝胶电泳(Native-polyacrylamide gel electrophoresis,Native-PAGE)分离获得个体化的体液免疫炎症相关蛋白复合物(immunoinflammation-related protein complexes,ⅡRPCs),ⅡRPCs水平在健康人与慢性疾病患者中存在显著差异。利用十二烷基磺酸钠聚丙烯酰胺凝胶电泳(Sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)从ⅡRPCs中分离得到的IgG,即为DSIgG。对DSIgG重链进行胶内酶解后,利用天然材料杨絮从酶解液中富集得到FcN-糖肽,再采用超高分辨率的基质辅助激光解吸电离-傅里叶变换离子回旋共振质谱仪(matrix-assisted laser desorption ionization-Fourier transform ion cyclotron resonance mass spectrometry,MALDI-FTICRMS)检测FcN-糖肽,统计分析不同病理状态下患者的糖肽差异,寻找可用于癌症诊断和病情进展监测的个体化标志物。本研究包括如下三个部分:1.DSIgG Fc N-糖基化修饰个体化标志物区分胃癌与胃部良性疾病。本部分共研究了 1037例胃部疾病患者,其中包括525例胃部良性疾病患者和512例胃癌患者,共检测到22种Fc N-糖肽,其中包括8种DSIgG1 N-糖肽(糖型为GOF、GOFN、G1、G1F、G1FN、G1S、G2F 和 G2FS)和 14 种 DSIgG2N-糖肽(糖型为 GOF、GOFN、G1、G1N、G1F、G1FN、G1S、G1FS、G2、G2S、G2N、G2F、G2FN和G2FS)。统计分析结果表明,胃部良性疾病与胃癌患者中具有G1、G1S、G2F、G1FN 糖型的 DSIgG1 N-糖肽和具有 GOF、GOFN、G1N、G1FS、G2、G2F、G2FN、G2FS糖型的DSIgG2N-糖肽具有显著差异,糖肽相对强度比以及糖基化修饰水平与病理状态相关。与胃部良性疾病患者相比,胃癌患者的唾液酸化修饰水平以及半乳糖基化修饰水平(特别是双半乳糖基化修饰水平)均显著降低,而平分型N-乙酰葡萄糖胺水平则显著升高。在女性中,相差一个半乳糖残基的糖肽相对强度比(G2FN/G1FN)可以显著区分胃部良性疾病与胃癌,受试者工作特征(Receiver operating characteristic,ROC)分析表明,曲线下面积(Area under the curve,AUC)为0.872,特异度和灵敏度均为82.6%。在男性中,相差一个核心岩藻糖残基的糖肽相对强度比(G2FN/G2N)和相差一个半乳糖残基的糖肽相对强度比(G2F/G1F)的组合可用于60岁以下男性人群的胃部良性疾病与胃癌的区分,其AUC为0.846。DSIgG2 G1FS糖型、G2FN/G2N比值和DSIgG1相差两个半乳糖残基的糖肽相对强度比(G2F/G0F)的组合可以区分60岁以上男性人群的胃部良性疾病与胃癌,其AUC为0.777。本部分研究结果表明,Fc N-糖基化修饰类型与患者胃部病理状态显著相关,可作为一类新型的胃癌个体化诊断的潜在标志物。2.DSIgG Fc N-糖基化修饰个体化标志物区分非小细胞肺癌与肺部良性疾病本部分共研究了 986例肺部疾病患者,其中包括509例肺部良性疾病患者和477例非小细胞肺癌患者,采用高分辨质谱技术分析了 DSIgG Fc N-糖基化修饰。统计分析结果表明,糖基化修饰水平与年龄相关。与肺部良性疾病患者相比,非小细胞肺癌患者的核心岩藻糖基化修饰水平和唾液酸化修饰水平显著升高,而半乳糖基化修饰水平则显著降低。ROC分析结果表明,相差一个核心岩藻糖残基的糖肽相对强度比(DSIgG2 G1F/G1G1F/G1、G1FS/G1S 和 DSIgG1 G1F/G1)的组合,可以区分 60岁以下年龄组肺部良性疾病与非小细胞肺癌,其AUC均优于0.76,灵敏度优于87%;而DSIgG2相差一个核心岩藻糖残基的糖肽相对强度比(G2F/G2、G1FS/G1S)与相差一个唾液酸残基的糖肽相对强度比(G1FS/G1F)的组合,可以区分60岁以上年龄组肺部良性疾病与非小细胞肺癌,其AUC均优于0.78,灵敏度优于91%。本部分研究表明,FcN-糖肽相对强度比水平与肺部病理状态显著相关,可作为一类新型的非小细胞肺癌个体化诊断的潜在标志物。3.DSIgG Fc N-糖基化修饰个体化标志物监测非小细胞肺癌疾病进展耐药性的产生可能造成非小细胞肺癌患者在靶向治疗过程中出现疾病进展,尽早发现疾病进展有助于在癌症复发甚至恶化早期及时采取有效的治疗措施,提高患者的生存率与生存质量。本部分采用高分辨质谱技术共检测36名非小细胞肺癌患者的413例随访血液样本中的DSIgG Fc N-糖肽。Kaplan-Meier曲线表明相差一个半乳糖残基的糖肽相对强度比(DSIgG1 G2F/G1F)以及相差一个核心岩藻糖残基的糖肽相对强度比(DSIgG2 G1F/G1)与患者的无疾病进展生存期显著相关。随访样本分析结果表明,利用DSIgG Fc N-糖基化修饰指标(DSIgG1 G1F/G1、G1S/G1和DSIgG2G1S/G1、G1FN/G1N、G1FN/G0FN)可以个体化的监测非小细胞肺癌的疾病进展时间点,比临床影像学检测的疾病进展时间点平均提前29周(四分位数区间:16-34周)。本研究结果表明,DSIgG FcN-糖基化修饰水平与非小细胞肺癌的疾病进程密切相关,应用DSIgG Fc N-糖基化修饰标志物有助于评估靶向治疗疗效和调整治疗策略,可作为个体化监测非小细胞肺癌患者疾病进展的潜在标志物。
[Abstract]:Cancer is the second largest killer of human health after cardiovascular disease, among which, gastric cancer and lung cancer are in the forefront of the incidence and mortality of cancer. The early symptoms of cancer patients, if misdiagnosed as related inflammation, may lead to missing the best time for treatment and reduce the chances of survival, so the diagnosis of benign diseases and cancer G (Immunoglobulin G (IgG) is one of the most important immune molecules in the human body. Its Fc N- glycosylation modification can change the interaction between IgG molecules and Fc receptors, thus affecting the proinflammatory or anti-inflammatory activity of IgG, and is significantly related to the physiological and pathological state of human beings. The study mainly focused on the total IgG of blood, which, to some extent, ignored the structural change of antigen specific IgG as the protein molecule in the peripheral blood only inferior to albumin, not all IgG molecules were associated with the development of the disease. The previous study showed that the antigen specific IgG glycosylation modification and blood in the process of disease development The variation trend of total IgG glycosylation modification is not completely consistent, so the study of antigen specific IgG or disease specific IgG (Disease-specific IgG, DSIgG) can more accurately reflect the pathological state. This study uses non denaturated acrylamide gel electrophoresis (Native-polyacrylamide gel electrophoresis, Native-PAGE) to separate the individual body fluid The immune inflammatory related protein complex (immunoinflammation-related protein complexes, II RPCs), and the level of II RPCs in healthy people and chronic disease patients were significantly different. Twelve sodium alkyl sulfonate polyacrylamide gel electrophoresis (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) was isolated from the second RPCs. The obtained IgG, that is, after the enzymatic hydrolysis of the DSIgG heavy chain by DSIgG., enriched the FcN- glycopeptide by the natural material poplar flocculation from the enzymolysis solution, and then used the ultra high resolution matrix assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometer (matrix-assisted laser desorption ionization-Fourier transform ion cyclotron). Resonance mass spectrometry, MALDI-FTICRMS) detection of FcN- glycopeptides, statistically analyzing the glycopeptide differences in patients with different pathological conditions, looking for individual markers that can be used for cancer diagnosis and progression monitoring. This study includes the following three parts: 1.DSIgG Fc N- glycosylated individualized markers distinguish gastric cancer and gastric benign disease. A total of 1037 patients with gastric diseases, including 525 patients with benign gastric diseases and 512 cases of gastric cancer, were studied, and 22 Fc N- glycopeptides were detected, including 8 DSIgG1 N- glycopeptides (GOF, GOFN, G1, G1F, G1FN, G1S, G2F and G2FS) and 14 kinds of glycopeptides N, G2F, G2FN and G2FS). The statistical analysis showed that the gastric benign disease and gastric cancer patients have G1, G1S, G2F, G1FN sugar DSIgG1 N- glycopeptides and GOF, the relative intensity ratio of glycosylated peptide and the level of glycosylated modification related to the pathological state. Compared with the patients with disease, the level of acidification modification and the level of galactoylation modification (especially the level of double galactoylation) in the patients with gastric cancer were significantly reduced, while the level of the N- acetyl glucosamine was significantly increased. In women, the relative intensity ratio of a galactopeptide with a different galactose residue (G2FN/G1FN) could significantly distinguish the stomach from the stomach. The Receiver operating characteristic (ROC) analysis showed that the area under the curve (Area under the curve, AUC) was 0.872, and the specificity and sensitivity were 82.6%. in men, and the relative intensity ratio (G2FN/G2N) and the difference of a galactose residue from a core fucose residue were different. The combination of peptide relative intensity ratio (G2F/G1F) can be used to distinguish between benign gastric disease and gastric cancer in male population under 60 years of age. The combination of AUC is 0.846.DSIgG2 G1FS sugar, and the relative intensity ratio (G2F/G0F) of two galactose residues of G2FN/G2N ratio and DSIgG1 can be divided into benign gastric disease and gastric cancer in male population over 60 years old. The results of its AUC 0.777. study show that the type of Fc N- glycosylation modification is significantly related to the pathological state of the stomach. It can be used as a potential marker for a new type of individualized diagnosis of gastric cancer,.2.DSIgG Fc N- glycosylated individualized markers to distinguish between non small cell lung cancer and benign lung diseases in this part, 986 cases of lung were studied. DSIgG Fc N- glycosylation modification was analyzed by high resolution mass spectrometry, including 509 patients with benign lung disease and 477 patients with non-small cell lung cancer. Statistical analysis showed that the glycosylation level was related to age. Compared with the benign lung disease patients, the core fucoylation of non small cell lung cancer patients was compared with those of the patients with benign lung disease. The level of modification and acidification of saliva increased significantly, while the level of galactin modification was significantly reduced by.ROC analysis. The relative intensity ratio of a core fucose residue (DSIgG2 G1F/G1G1F/G1, G1FS/G1S and DSIgG1 G1F/G1) could be divided into benign and non small lung diseases in the age group under 60 years of age. The AUC of cell lung cancer is better than 0.76 and the sensitivity is better than 87%, while the relative intensity of the glycopeptide relative intensity of a core fucose residue (G2F/G2, G1FS/G1S) and the difference of the relative intensity ratio (G1FS/G1F) of one sialic acid residue (G1FS/G1F) can distinguish between the 60 years old age group and the non small cell lung cancer in the age group over 60 years old, and the AUC is better than 0.78. The sensitivity is better than the 91%. part of this study. The relative intensity ratio of FcN- glycopeptide is significantly related to the pulmonary pathological state. It can be used as a new type of potential marker for individual diagnosis of non-small cell lung cancer,.3.DSIgG Fc N- glycosylated individualized marker monitoring the development of non small cell lung cancer disease development resistance may cause non Patients with small cell lung cancer have developed disease in the course of targeted therapy. The early discovery of disease progress helps to take effective treatment measures in the early stage of cancer recurrence and even worsening, and improves the survival rate and quality of life of the patients. This part uses high resolution mass spectrometry to detect 413 cases of 36 patients with non-small cell lung cancer. The DSIgG Fc N- glycopeptide.Kaplan-Meier curve in the sample indicates that the relative intensity ratio of the glycopeptide relative intensity ratio (DSIgG1 G2F/G1F) and the difference of a core fucose residue (DSIgG2 G1F/G1) is significantly related to the patient's disease progression survival period (DSIgG2 G1F/G1). The follow-up sample analysis shows that DSIgG Fc N- sugar is used. DSIgG1 G1F/G1, G1S/G1 and DSIgG2G1S/G1, G1FN/G1N, G1FN/G0FN) can monitor the progression of non small cell lung cancer at a time point, 29 weeks earlier than the time point of the disease progression by clinical imaging (four quantile interval: 16-34 weeks). The results of this study showed that the level of glycosylation of DSIgG FcN- and non small The process of cell lung cancer is closely related. The application of DSIgG Fc N- glycosylation markers can help to evaluate the therapeutic effect and adjust the therapeutic strategy, which can be used as a potential marker for individual monitoring of the disease progression in non small cell lung cancer patients.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.2;R734.2

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