【作者】 马维东；

【导师】 郝希山； 李强；

【作者基本信息】 天津医科大学 ， 肿瘤学， 2003， 博士

【摘要】 原发性肝癌是严重威胁人类健康的恶性肿瘤，尤其是在亚洲国家发病率高。目前肝癌的治疗手段仍主要依赖于外科手术切除，而放疗和化疗对肝癌的治疗效果很差，肝癌总体的治疗效果仍不能令人满意。影响肝癌远期疗效的主要问题是术后复发和远处转移。对于术后复发和转移或由于多种原因无法手术的患者，以肿瘤抗原为基础的特异性抗肿瘤免疫治疗作为新兴的治疗手段有望改善这部分患者的治疗效果。 自20世纪50年代Foley和Prehn等人在同系小鼠的研究中发现了肿瘤特异性抗原，近20年基础免疫学已经获得了巨大的发展。特别是各种肿瘤特异性抗原的不断发现，以及对免疫应答机制的深入了解，针对各种肿瘤特异性抗原表位的疫苗备受关注并进行了大量的基础研究及临床试验。其中肿瘤／睾丸抗原(cancer／testis,CT)以其严格的肿瘤特异性和良好的免疫原性成为其中的代表。 CT抗原在多种恶性肿瘤中表达，目前国内外关于CT抗原的研究大多集中在恶性黑色素瘤、乳腺癌、肺癌、和卵巢癌，在肝癌中的表达研究少见报道。我们选择了CT抗原中最具代表性的MAGE-3和NY-ESO-1，研究了这两种CT抗原的mRNA在肝癌中的表达，并对其与病例的临床病理资料以及预后等因素的关系进行了分析。进一步研究人工合成的MAGE-3和NY-ESO-1短肽疫苗负载成熟DC(dendritic cell,树突状细胞)，在体外诱导特异性CTL(cytotoxic T天津医科大学博士学位论文lymphocyte，细胞毒性T细胞)对人工负载同一抗原表位以及天然表达MAGE一3及NY一ESO一1抗原的靶细胞的杀伤作用。 第一部分 肿瘤特异性抗原MAGE一3和NY-ESO一1在肝癌组织中的表达 及临床病理分析 目的:研究肿瘤特异性抗原和NY-ESO一1在肝癌组织中的表达，了解表达阳性与临床病理特征及预后的关系。方法:选择66例肝癌手术切除标本，用RT一PCR方法检测MAGE一3及NY一ESO一lmRNA的表达。结果:54例HCC的MAGE一3表达阳性率为64.81%;其中的25例HCC中NY一ESO一1阳性率为32%，9例转移性肝癌中1例乳腺癌肝转移MAGE一3阳性。MAGE一3及NY一Eso一1 mRNA的表达与临床病理及预后无关(p<0.05)。结论:MAGE一3及NY一ESO一lmRNA在HCC高表达，但与恶性黑色素瘤、卵巢癌等不同，其表达的阳性不提示预后。第二部分 树突状细胞的体外培养及功能鉴定 目的:体外培养DC作为人工合成肚疫苗的抗原递呈细胞。方法:用贴壁的外周血单个核细胞(peripheral blood mononuclear cell，pBMC)在含有粒细胞巨噬细胞集落刺激因子(granulocyte一macrophage eolo叮stimulatingfactor，GM一CSF)和白细胞介素一4(IL一4)的1640培养基体外培养成DC，用流式细胞仪鉴定表型，并进行功能检测。结果:外周血单个核细胞能够诱导成成熟DC，并具有生物学活性。结论:经实验证实PBMC体外培养的成熟DC具有生物学活性，可以作为人工合成肤疫苗的有效载体。天津医科大学博士学位论文第三部分人工合成的MAGE一3及NY-ESO一1抗原肤体外诱导CTL 对相应靶细胞的杀伤作用 目的:研究人工合成的MAGE一3及NY一ESO一1抗原肤诱导自体T细胞产生CTL对相应靶细胞的特异性杀伤作用。方法:人工合成的MAGE一3及NY一ESO一1抗原肤负载成熟DC后刺激自体淋巴细胞诱导产生特异性CTL，LDH (乳酸脱氢酶)法对人工负载同一抗原及天然表达相应抗原的靶细胞进行杀伤试验。结果:人工合成的MAGE一3及NY一ESO一1抗原肤负载成熟DC后刺激自体淋巴细胞能够诱导产生特异性CTL，并能够对负载同一抗原肤的靶细胞产生特异性杀伤作用。结论:人工合成的抗原短肤可以诱导产生特异性的CTL，可以作为候选的肿瘤肤疫苗。更多还原

【Abstract】 Liver cancer is one of the most detrimental cancer and with a relatively high morbidity in Asian country. Now the treatment of liver cancer is mainly dependent on surgical resection. Despite the rapid development of hepatectomy and the improvement on the 5-year-surval rate of small cancer with a diameter less than 3 centimeter in the past 50 years, the outcome of liver cancer is unsatisfactory. The poor prognosis is mainly due to recurrence and metastasis post-operation. For these patients and those who can not take the operation for different reasons, immunotherapy rise the hope to improve the treatment.Since the discovery of tumor specific antigen by Foley and his collegers in 50’s of 20 century, the development of basic immunology is more rapidly in past 20 years, especially on the identification of different tumor specific antigen and well understanding of the mechanism of immune response. Many research work and clinical trials on tumor vaccines on the base of epitopes about tumor antigen have been conducted, and cancer/testis (CT) antigens are pay much more attention for their best tumor specific and high immunogenicity.CT antigens are expressed in variety cancers, include melanoma, breast cancer, lung cancer, ovarian cancer but few report about hepatocellular carcinoma. We study the expression of two CT antigen MAGE-3 and NY-ESO-1 in hepatocellular carcinoma, and analyzed the clinical features. Further more we usesynthetic peptide coding by the epitope of these antigens and load on DC to induce special CTL witch lyses the target cells loading with the same antigen.Part I Study on the expression of MAGE-3 and NY-ESO-1 in humanHCC and analysis with clinical featuresObjective: To investigate the expression of cancer/testis antigen MAGE-3 and NY-ESO-1 in human hepatocellular carcinoma and evaluate the positive expression with the clinical features. Method: 60 specimen by hepatectomy with liver cancer were detected using RT-PCR. Results: MAGE-3 mRNA were detected in 64.81% of 54 HCC, and NY-ESO-1 in 32% of 25 HCC. The expression of such genes has no responding with clinical-pathological features. Conclusion:MAGE-3 and NY-ESO-1 express highly in HCC and the expression of these genes can not indicate the prognosis.Part II .Culture and function examination ofdendritic cells in vitroObjective: culture the dendritic cells in vitro as antigen presenting cell of peptide vaccine. Method: Culture the PBMC with 1640 containing GM-CSF and IL-4,identify the phenotype with flow cytometry and exam the bioactivity with IL-12/INF-r secretion. Results: DC can be cultured from monocyte and has the bioactivity. Conclusion: mature DC can be cultured from monocyte and use as vector of peptide vaccine.Part lH.Synthetic peptide coding with the epitope of MAGE-3and NY-ESO-1 to induce special CTL in vitroObjective: To evaluate the synthetic peptide coding with the epitope of MAGE-3 and NY-ESO-1 inducing special CTL in vitro. Method: Mature DC loading with the synthetic peptide coding with the epitope of MAGE-3 and NY-ESO-1 to stimulate autologous T cells in order to induce special CTL and detect the special lysis of corresponding target cells by LDH. Results: synthetic peptide can induce special CTL and lysis the corresponding target cells bearing the same epitope. Conclusion: Synthetic peptide coding with the epitope of MAGE-3 and NY-ESO-1 can induce special CTL and take as the candidate of anti-tumor peptide vaccine.更多还原