个体化HLA-A2限制性肿瘤抗原肽的筛选方法建立及其在结肠癌抗原肽中的研究
发布时间：2019-06-29 19:34
【摘要】：近几年免疫检查点抗体治疗取得了巨大成功,但仍有超过50%的肿瘤患者未能对阻断治疗作出反应。同时,精准医学依靠验证过的生物标志物来更好地预测及划分患者可能的疾病风险,从而预先对治疗做出反应,这使寻找可靠的肿瘤抗原肽变得尤为重要。新技术(如第二代测序)的出现为肿瘤免疫学寻找新的免疫靶标提供了新的手段,使免疫治疗广泛地应用到肿瘤患者。在本论文中,将重点讨论肿瘤免疫治疗中肿瘤抗原肽的鉴定。恶性肿瘤自身抗原的表达可以激发肿瘤的免疫反应,肿瘤抗原肽特异性的免疫治疗可以推动肿瘤的消失。但是,目前鉴定出的肿瘤抗原肽特异性免疫治疗效果并不理想,尚需获得更有效的自身肿瘤抗原进行临床试验,使利用肿瘤疫苗或免疫疗法治愈肿瘤成为可能。本研究使用一种联合色谱、质谱及生物信息学预测分析的生化方法,以结肠癌肿瘤组织为研究对象,从免疫亲和纯化过的人类白细胞抗原-A2(Human leukocyte antigen-A2,HLA-A2)分子中获得12条结肠癌抗原肽即为CC1至CC12。由于无法大量获得天然的抗原肽,进而采取化学合成的方法,并测试它们是否可用于稳定细胞表面产生的主要组织相容性复合体(Major histocompatibility complex,MHC)分子,并分析其引起细胞毒性T淋巴细胞(Cytotoxic lymphocyte,CTL)免疫反应的能力。结果表明,该筛选抗原肽的方法有效。在混合肽检测结果中,混合肽(CC1、CC2、CC3、CC4)相对于空白对照组有显著性差异,可以认为CC1-4中至少有一个多肽是此肿瘤病人的结肠癌抗原肽。在单个肽检测结果中,CC1、CC9、CC10、CC11、CC12产生的γ-干扰素(Interferon-γ,IFN-γ)量皆有一定幅度的增加,CC2、CC5、CC6、CC7、CC8产生的IFN-γ量并无明显差异。基于混合肽中,仅有CC1-4有明显杀伤结果,推测此方法筛选出的结肠癌抗原肽CC1为此患者的结肠癌抗原肽,其余的CC9、CC10、CC11、CC12可能杀伤效果并不突出或者此检测方法并没有反映出他们的实际杀伤效果,有待后续试验从多角度验证。本论文叙述的课题也成功地建立了一种个体化HLA-A2限制性肿瘤抗原肽的筛选方法平台并应用在结肠癌抗原肽的研究。
[Abstract]:In recent years, immunocheckpoint antibody therapy has achieved great success, but more than 50% of tumor patients still fail to respond to blocking treatment. At the same time, precision medicine relies on verified biomarkers to better predict and divide the possible disease risk of patients, so as to respond to treatment in advance, which makes it particularly important to find reliable tumor antigen peptides. The emergence of new techniques, such as second generation sequencing, provides a new means for tumor immunology to find new immune targets, and makes immunotherapy widely used in tumor patients. In this paper, the identification of tumor antigenic peptides in tumor immunotherapy will be discussed. The expression of tumor autoantigen can stimulate the immune response of tumor, and the specific immunotherapy of tumor antigenic peptide can promote the disappearance of tumor. However, the specific immunotherapy effect of tumor antigenic peptides identified at present is not ideal, so it is necessary to obtain more effective self-tumor antigens for clinical trials to make it possible to use tumor vaccines or immunotherapy to cure tumors. In this study, 12 colon cancer antigenic peptides, CC1 to CC12., were obtained from human leukocyte antigen A2 (Human leukocyte antigen-A2,HLA-A2 purified by immunoaffinity using a biochemical method of combined chromatography, mass spectrometry and bioinformatics prediction and analysis. Due to the inability to obtain a large number of natural antigenic peptides, chemical synthesis methods were adopted to test whether they could be used to stabilize the major histochemical complex (Major histocompatibility complex,MHC produced on the surface of cells, and their ability to induce the immune response of cytotoxicity T lymphocytes (Cytotoxic lymphocyte,CTL) was analyzed. The results showed that the method of screening antigenic peptides was effective. In the results of mixed peptide detection, there was significant difference between mixed peptide (CC1,CC2,CC3,CC4) and blank control group. It can be considered that at least one polypeptide in CC1-4 is the antigenic peptide of colon cancer in patients with this tumor. In the results of single peptide detection, the amount of Interferon- 纬 (IFN- 纬) produced by CC1,CC9,CC10,CC11,CC12 increased to a certain extent, but there was no significant difference in the amount of IFN- 纬 produced by CC2,CC5,CC6,CC7,CC8. Based on the fact that only CC1-4 has obvious killing results, it is speculated that the colon cancer antigenic peptide CC1 screened by this method is the colon cancer antigenic peptide of the patients, and the other CC9,CC10,CC11,CC12 may not have outstanding killing effect or the detection method does not reflect their actual killing effect, which needs to be verified from many angles. In this paper, a platform for screening individual HLA-A2 restricted tumor antigenic peptides was also successfully established and applied to the study of colon cancer antigenic peptides.
【学位授予单位】：浙江理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R730.51
本文编号：2508053
[Abstract]:In recent years, immunocheckpoint antibody therapy has achieved great success, but more than 50% of tumor patients still fail to respond to blocking treatment. At the same time, precision medicine relies on verified biomarkers to better predict and divide the possible disease risk of patients, so as to respond to treatment in advance, which makes it particularly important to find reliable tumor antigen peptides. The emergence of new techniques, such as second generation sequencing, provides a new means for tumor immunology to find new immune targets, and makes immunotherapy widely used in tumor patients. In this paper, the identification of tumor antigenic peptides in tumor immunotherapy will be discussed. The expression of tumor autoantigen can stimulate the immune response of tumor, and the specific immunotherapy of tumor antigenic peptide can promote the disappearance of tumor. However, the specific immunotherapy effect of tumor antigenic peptides identified at present is not ideal, so it is necessary to obtain more effective self-tumor antigens for clinical trials to make it possible to use tumor vaccines or immunotherapy to cure tumors. In this study, 12 colon cancer antigenic peptides, CC1 to CC12., were obtained from human leukocyte antigen A2 (Human leukocyte antigen-A2,HLA-A2 purified by immunoaffinity using a biochemical method of combined chromatography, mass spectrometry and bioinformatics prediction and analysis. Due to the inability to obtain a large number of natural antigenic peptides, chemical synthesis methods were adopted to test whether they could be used to stabilize the major histochemical complex (Major histocompatibility complex,MHC produced on the surface of cells, and their ability to induce the immune response of cytotoxicity T lymphocytes (Cytotoxic lymphocyte,CTL) was analyzed. The results showed that the method of screening antigenic peptides was effective. In the results of mixed peptide detection, there was significant difference between mixed peptide (CC1,CC2,CC3,CC4) and blank control group. It can be considered that at least one polypeptide in CC1-4 is the antigenic peptide of colon cancer in patients with this tumor. In the results of single peptide detection, the amount of Interferon- 纬 (IFN- 纬) produced by CC1,CC9,CC10,CC11,CC12 increased to a certain extent, but there was no significant difference in the amount of IFN- 纬 produced by CC2,CC5,CC6,CC7,CC8. Based on the fact that only CC1-4 has obvious killing results, it is speculated that the colon cancer antigenic peptide CC1 screened by this method is the colon cancer antigenic peptide of the patients, and the other CC9,CC10,CC11,CC12 may not have outstanding killing effect or the detection method does not reflect their actual killing effect, which needs to be verified from many angles. In this paper, a platform for screening individual HLA-A2 restricted tumor antigenic peptides was also successfully established and applied to the study of colon cancer antigenic peptides.
【学位授予单位】：浙江理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R730.51
【参考文献】
相关期刊论文 前1条
1 王晓燕;分泌细胞因子的CD8~+T细胞亚群研究进展[J];国外医学(免疫学分册);1997年04期
，本文编号：2508053
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