【作者】 李林；

【导师】 金伯泉；

【作者基本信息】 第四军医大学 ， 免疫学， 2004， 博士

【摘要】 黑色素瘤抗原(melanoma antigen，MAGE)基因是首先从恶性黑色素瘤中分离、鉴定出的一族肿瘤抗原基因，这类抗原在正常组织中除了睾丸和胎盘组织外均不表达，但在某些肿瘤组织中呈高度特异性表达，部分家族成员可以诱导机体产生特异性的CTL细胞，因而在肿瘤的免疫学治疗方面具有潜在的应用价值。 MAGE-A10属于MAGE-A亚家族，其cDNA全长2.5kb，含有4个外显子，编码蛋白约72kDa。MAGE-A10在肿瘤细胞有较高水平表达，其具有提高肿瘤免疫原性的特点已受到广泛关注。新近采用四聚体(Tetramer)技术发现其编码的HLA-A2限制的九肽GLYDGMEHL(254-262位)可以诱导特异性的CTL应答。基于亚洲人多表达HLA—A2的特点，进一步研究MAGE-A10的表达、功能和潜在的肿瘤治疗作用显得尤为重要。为了研究MAGE-A10蛋白在肿瘤组织的表达规律，进一步了解其功能，探讨其在肿瘤免疫治疗中可能的作用，我们开展了如下工作： 1、人黑色素瘤抗原MAGE-A10的基因克隆与原核表达 目的：克隆人黑色素瘤抗原MAGE-A10(melanoma antigen A10)cDNA，构建原核表达载体，获得大肠杆菌表达的融合蛋白以用于单克隆抗体的制备。方法：从人黑色素瘤细胞系LiBr中提取总RNA，用RT-PCR从中扩增出MAGE-A10 cDNA，插入载体pMD18-T中。测序正确后，克隆至原核表达载体pGEX-4T-3中， 构建重组表达载体DGEX-4T-3-MAGE-A10，转化大肠杆菌BL21株进行表达。经IPTG诱 第四军医大学博士学位论文导表达、谷耽甘肤亲和层析获得重组目的蛋白。结果:成功获得MAGE一A10编码基因并构建原核表达载体，测序结果与GenBank收录序列相一致。可以获得部分可溶性的原核重组蛋白，经亲和层析分析，证实融合蛋白占总量的58.9%。SDS一PAGE分析其相对分子质量(阿r)为67KD，WesternBI0t证实为目的蛋白，为制备MAGE一A10单抗及研究MAGE一A10可能参与肿瘤发生发展的机制奠定了基础。 2、人黑色素瘤抗原MAGE一A10特异性单抗的制备与鉴定 目的:制备MAGE一A10特异性单抗并进行鉴定。方法:大量制备MAGE一AIO一GST融合蛋白，纯化后免疫BALB/c小鼠，应用杂交瘤技术制备了抗MAGE一A10的单克隆抗体。在此基础上，用正常翠丸组织的免疫组化染色进行了筛选和鉴定，采用硫酸按盐析、SepharoseG一25柱除盐和FpLC Q Fast Flow阴离子交换柱连续梯度洗脱的方法纯化抗体，重复免疫组化染色确定下一步试验用的抗体。结果:制备获得了19株抗MAGE一A10的单克隆抗体，遴选出一株高效、特异的单抗以一MAGE一AIO.6。采用上述纯化方法，获得了90%以上纯度的单克隆抗体。重复试验表明，经该种方法纯化后可以得到高纯度和高活性的单克隆抗体，为开展MAGE一A10表达分布的研究及探索其参与肿瘤发生发展的可能机制提供了工具。 3、MAGE一A10蛋白在多种肿瘤组织中的表达分布研究 目的:以制备的抗MAGE一A10单抗LX一MAGE一A10.6开展的免疫组化S一p染色(Immunohistoehemistry，IHC)，结合组织芯片分析 (TISSue Array，TA)，观测MAGE一A10蛋白在肺癌和四种不同消化系统肿瘤的表达水平;重点分析了其与肺癌病理类型、病理分级的关系。结果:①MAGE一A10蛋白在小细胞肺癌、肺鳞癌和肺腺癌中表达的阳性率分别为36.4%、76.5%和40.0%，在大细胞肺癌和支气管肺泡癌中未见阳性表达。MAGE一A10蛋白在小细胞肺癌与非小细胞肺癌的表达率未见差异，其与肺鳞癌、肺腺癌的病理分级也无明显相关。②MAGE一A10蛋白在食管鳞癌、肝细胞癌、胃癌和结肠癌均有表达，其中在食管癌中表达比例最高(73.3%)，主要在核内表达，而在肝细胞癌、胃癌和结肠癌中表达水平较低，比例分别为38.1%、15.4%和10.6%。结论:州AGE一A10蛋白在肺鳞癌中表达率最高，肺腺癌和小细胞肺癌次之，大细胞肺癌和支 第四军医大学博士学位论文气管肺泡癌中未见表达，此研究结果对不同病理类型肺癌的鉴别诊断可能有一定的辅助诊断意义，并可能成为某些肺癌如肺鳞癌生物治疗的潜在靶点。MAGE一A10蛋白在四种消化系统恶性肿瘤中均有表达，以鳞癌多见，呈异质性表达特点。 4、PEGFP·Cl一MAGE一A10真核表达载体和PLNCX一MAGE一A10逆转录病毒表达载体的构建与初步应用 目的:构建MAGE一AIO功能研究的必要的工具载体，并观察MAGE一A10的细胞内定位。方法:利用基因克隆技术，在前期获得MAGE一A10开放读框的基础上，构建pEG「p一Cl一MAGE一AIO真核表达载体和pLNCX一MAGE一AIO逆转录病毒表达载体。将pEGFp一Cl一MAGE一A10转入293丁细胞，观察MAGE一A10的亚细胞定位情况。结果:成功构建pEGFp一Cl一MAGE一A10真核表达载体和pLNCX一MAG一A10逆转录病毒表达载体，发现MAGE一AIO蛋白主要定位细胞核内。更多还原

【Abstract】 Melanoma antigen genes (MAGEs) were a number of gene families cloned from Melanoma, which expressed specifically on some types of cancers but not on normal tissue except for testis and placenta. Most members of MAGE families can induce specific CTL response in host, so they have potential value in the immunotherapy of tumors.MAGE-A10 belongs to MAGE-A subfamily. The full length of cDNA was 2.5kb contained 4 exons, coding a protein with Mr of 72kDa. MAGE-A 10 was highly expressed on tumor cells with a high immunogenesity. A HLA-A2 restricted nona-peptide 254GLYDGMEHL262 coded by MAGE-A10 could elicit specific CTL response which was found by tetramer technology recently. Based on highly frequency of HLA-A2 in Asians, it is important to investigate the expression, function and potential immunotherapy value of this molecule. To explore the expression of MAGE-A 10 and distribution of that in cancers, the following works have been done in our department:1. Cloning and prokaryotic expression of human MAGE-A10 cDNAAims: To clone human MAGE-A10 cDNA, construct the recombination expression vector, and express its recombinant protein in E. coli. Methods: The cDNA encoding human MAGE-A 10 gene was amplified by RT-PCR from human melanoma cell line LiBr. Then the MAGE-A 10 gene was inserted into plasmids pMD18-T. After sequencing, the MAGE-A 10 was cloned into the prokaryotic expression vector pGEX-4T-3 to construct the recombinantexpression vector pGEX-4T-3-MAGE-A10 and were transformed into E. coli BL21.The recombinant GST-MAGE-A10 fusion protein was expressed under induction of IPTG. GST-MAGE-A10 fusion protein was purified through glutathione agarose column. Results: The sequence of MAGE-AIO was identical to that from GenBank. Affinity column purified-GST-MAGE-A10 fusion protein appeared a band of Mr. 70KD on SDS-PAGE, and the antigenic specificity of the fusion protein was confirmed by Western blot. Conclusions: The MAGE-A10 gene was cloned and expressed successfully, which not only provided the immunogen for preparation of anti-MAGE-A10 antibody but also were useful in research work on the mechanism of tumor pathogenesis and cellular immunological response to MAGE molecules.2. Preparation and identification of monoclonal antibodies against human MAGE-AIOAims: To prepare and identify the monoclonal antibody to MAGE-A10 Methods: GST-MAGE-A10 fusion protein was largely prepared and purified. Then BALB/c mice were immunized with purified-GST-MAGE-A10 fusion protein to get anti-MAGE-A10 antibody. Normal testis tissue was used to identify specific mAbs. The anti-MAGE-A10 antibody was purified as follows:Firstly, mAbs from ascites fluid were sedimentated with ammonium sulfate,then desalted by Sepharose G-25 column and purified by anion exchange chromatography on Sepharose Q Fast Flow column using FPLC (Phamarcia Amercia). Results: 19 hybridoma cell lines secreting mAbs against MAGE-A10 have been established. One of them named LX-MAGE-A10.6 was employed in immunohistochemical staining with highly specific and sensitivity. Conclusions: We got one hybridoma cell which could be useful for investigation of its expression and distribution.3. The expression patterns of MAGE-A10 in tumorsAims: To explore the expression and the clinical significance of MAGE-A10 in human lung cancer and other four types of digestive system cancers. Methods: By using tissue array and S-P immunohistochemical methods, the expression of MAGE-A10 protein in lung cancer and four types of digestive system cancers was investigated and the correlation of MAGE-A10 expression with histological types and pathological grades was also evaluated. Results: (1)The positive expression rates of MAGE-A10 were 36.4% in small cell lung carcinoma(SCLC), 76.5% in squamous cell lung carcinoma and 40.0%in lung adenocarcinoma, respectively, whereas no positive case can be found in large cell lung carcinoma and bronchioloalveolar carcinoma. There was no statistical significance between the expression of MAGE-A10 protein in SCLC and that in NSCLC. The更多还原