個(gè)體化HLA-A2限制性腫瘤抗原肽的篩選方法建立及其在結(jié)腸癌抗原肽中的研究
發(fā)布時(shí)間：2019-06-29 19:34
【摘要】：近幾年免疫檢查點(diǎn)抗體治療取得了巨大成功,但仍有超過(guò)50%的腫瘤患者未能對(duì)阻斷治療作出反應(yīng)。同時(shí),精準(zhǔn)醫(yī)學(xué)依靠驗(yàn)證過(guò)的生物標(biāo)志物來(lái)更好地預(yù)測(cè)及劃分患者可能的疾病風(fēng)險(xiǎn),從而預(yù)先對(duì)治療做出反應(yīng),這使尋找可靠的腫瘤抗原肽變得尤為重要。新技術(shù)(如第二代測(cè)序)的出現(xiàn)為腫瘤免疫學(xué)尋找新的免疫靶標(biāo)提供了新的手段,使免疫治療廣泛地應(yīng)用到腫瘤患者。在本論文中,將重點(diǎn)討論腫瘤免疫治療中腫瘤抗原肽的鑒定。惡性腫瘤自身抗原的表達(dá)可以激發(fā)腫瘤的免疫反應(yīng),腫瘤抗原肽特異性的免疫治療可以推動(dòng)腫瘤的消失。但是,目前鑒定出的腫瘤抗原肽特異性免疫治療效果并不理想,尚需獲得更有效的自身腫瘤抗原進(jìn)行臨床試驗(yàn),使利用腫瘤疫苗或免疫療法治愈腫瘤成為可能。本研究使用一種聯(lián)合色譜、質(zhì)譜及生物信息學(xué)預(yù)測(cè)分析的生化方法,以結(jié)腸癌腫瘤組織為研究對(duì)象,從免疫親和純化過(guò)的人類白細(xì)胞抗原-A2(Human leukocyte antigen-A2,HLA-A2)分子中獲得12條結(jié)腸癌抗原肽即為CC1至CC12。由于無(wú)法大量獲得天然的抗原肽,進(jìn)而采取化學(xué)合成的方法,并測(cè)試它們是否可用于穩(wěn)定細(xì)胞表面產(chǎn)生的主要組織相容性復(fù)合體(Major histocompatibility complex,MHC)分子,并分析其引起細(xì)胞毒性T淋巴細(xì)胞(Cytotoxic lymphocyte,CTL)免疫反應(yīng)的能力。結(jié)果表明,該篩選抗原肽的方法有效。在混合肽檢測(cè)結(jié)果中,混合肽(CC1、CC2、CC3、CC4)相對(duì)于空白對(duì)照組有顯著性差異,可以認(rèn)為CC1-4中至少有一個(gè)多肽是此腫瘤病人的結(jié)腸癌抗原肽。在單個(gè)肽檢測(cè)結(jié)果中,CC1、CC9、CC10、CC11、CC12產(chǎn)生的γ-干擾素(Interferon-γ,IFN-γ)量皆有一定幅度的增加,CC2、CC5、CC6、CC7、CC8產(chǎn)生的IFN-γ量并無(wú)明顯差異�；诨旌想闹�,僅有CC1-4有明顯殺傷結(jié)果,推測(cè)此方法篩選出的結(jié)腸癌抗原肽CC1為此患者的結(jié)腸癌抗原肽,其余的CC9、CC10、CC11、CC12可能殺傷效果并不突出或者此檢測(cè)方法并沒(méi)有反映出他們的實(shí)際殺傷效果,有待后續(xù)試驗(yàn)從多角度驗(yàn)證。本論文敘述的課題也成功地建立了一種個(gè)體化HLA-A2限制性腫瘤抗原肽的篩選方法平臺(tái)并應(yīng)用在結(jié)腸癌抗原肽的研究。
[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- 緯 (IFN- 緯) produced by CC1,CC9,CC10,CC11,CC12 increased to a certain extent, but there was no significant difference in the amount of IFN- 緯 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.
【學(xué)位授予單位】：浙江理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R730.51
本文編號(hào)：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- 緯 (IFN- 緯) produced by CC1,CC9,CC10,CC11,CC12 increased to a certain extent, but there was no significant difference in the amount of IFN- 緯 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.
【學(xué)位授予單位】：浙江理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R730.51
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 王曉燕;分泌細(xì)胞因子的CD8~+T細(xì)胞亞群研究進(jìn)展[J];國(guó)外醫(yī)學(xué)(免疫學(xué)分冊(cè));1997年04期
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