【作者】 徐璐；

【导师】 冯振卿；

【作者基本信息】 南京医科大学 ， 病理学， 2005， 硕士

【摘要】 肝细胞癌(hepatocellular carcinoma,HCC)在我国发病率高,临床疗效及预后均很不理想,近来针对肝细胞癌的免疫治疗受到瞩目。但是长期以来,人们未能在肝癌中发现适用于诱导保护性免疫应答的肿瘤抗原,因而阻碍了肝癌免疫治疗的进展。MAGE-A(melanoma antigen A)基因是首先从黑色素瘤中发现的一组肿瘤相关抗原,这类抗原在正常组织中除睾丸和胎盘外均不表达,但在某些恶性肿瘤高度特异性表达,同时经MHC-Ⅰ类分子递呈到细胞表面后,可为自体细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)识别,因而是一种CTL介导的特异性免疫治疗的理想靶分子。现已知MAGE-A基因家族成员达12个,各成员间的同源性较高。MAGE-A9 cDNA全长945bp,编码分子量约35Kd的蛋白产物。迄今未见关于MAGE-A9基因克隆表达及在肝细胞癌中定位的研究报道。本课题在参照国内外相关研究的基础上,设计并完成了以下研究内容: 1.从HCC组织中抽提总RNA,设计引物扩增MAGE-A9基因,经亚克隆、测序鉴定其序列,与GenBank数据库比对。 2.构建MAGE-A9基因的原核分泌表达系统,分离可溶性表达产物,鉴定并纯化目的蛋白。 3.制备抗MAGE-A9抗原的单克隆抗体。 4.观察MAGE-A9基因在HCC中的表达及定位。 研究方法 1.Trizol一步法提取肝细胞癌组织总RNA,RT-PCR扩增更多还原

【Abstract】 The occurrence of HCC is high in China. However, there has no any satisfied treatment against hepatocellular carcinoma. A potentially useful strategy for treating HCC is to develop vaccines against antigens that are commonly expressed on this type of cancer. It has been reported that the specific expression of some MAGE-A genes in HCC is high. Therefore, the tumor-specific expression has made the MAGE-A genes interesting as candidates for anti hepatocellular carcinoma immunotherapy.In 1991, van der Bruggen et al. identified the first melanoma antigen gene, MAGE-A1, in a human melanoma cell line. Since then, 12 MAGE-A genes have been isolated and sequence homology. Genes of MAGE-A family are expressed in several types of solid tumors but are silent in normal tissues with the exception of male germline cells. Therefore, peptides encoded by MAGE-A genes are strictly tumor-specific antigens that can be recognized by CTL and constitute promising targets for diagnosis and therapy. MAGE-A9 is of particular relevance in the context of the MAGE-A gene family, which has been mapped to chromosome X, and its gene product has been identified as a protein of an apparent molecular weight of Mr 35,000. Since little is known about the expression of MAGE-A9 in hepatocellular carcinomas in Chinese population, we examined the expression of MAGE-A9 in 39 prognostic parameters of hepatocellular carcinoma. In this study, we fulfilled some objects. Objectives:1. To extract total RNA of MAGE-A9 gene from hepatocelllar carcinoma tissue, to amplify the MAGE-A9 cDNA products and to subclone it into the T-vector for DNA sequencing analysis and identifying the sequence.2. To construct the prokaryotic expression system of MAGE-A9 gene, to identify and purify the culture products.3. To generate the monoclonal antibody of MAGE-A9 antigen4. To examine the expression and location of MAGE-A9 in HCCMethods1. Total RNA was extracted from the hepatocellular carcinoma tissue by using Trizaol and reverse transcribed. MAGE-A9 cDNA was amplified by PCR, ligated with a similarly restricted pMD-18T simple vector, sequceced and identified after comparing with GenBank.2. The purified plasmid was digested with EcoR Ⅰ and Hind Ⅲ and subcloned into expression plasmid vector pBAD/gⅢ with six-histidine tag and the recombinant plasmid was transferred into E.coli TOP 10. This Culture was induced by L-arabinose and pufified by HiTrap affinity columns. SDS-PAGE and Western blot monitored the production and purification of the recombinant protein.3. BALB/c mice were immunized with purified material. Animals were sacrificed, and fusions were performed as described in manuscript. Hybridoma supernatants were screened by ELISA and the purified MAGE-A9 protein was used as the antigen. The monoclonal antibody was identified by SDS-PAGE and Dot blot.4. The wxpression and location of MAGE-A9 in HCC were examined through Immunohistochemical assay.Results1. The 945bp MAGE-A9 gene was amplified and its sequence product was same to MAGE-A9 cDNA published on GenBank2. Restriction mapping of recombinant plasmid revealed that the cloning vector pMD18-T - MAGE-A9 was successfully constructed. L-arabinose induction of the expression system pBAD/gIII-MAGE-A9 resulted in the production of a protein exhibiting an apparent Mr 35,000, matching the previously described MAGE-A9 gene product and identified by Western blot. Purified products were generated by using HiTrap column.3. The positive rate of ELISA was 100% after three subclones and a stable hybridoma cell line that cans secret anti-MAGE-A9 monoclonal antibody has been created and then been named AM9. The results of Dot blot revealed that the cell line secreting anti-MAGE-A9 monoclonal antibody showed exclusive reactivity on the MAGE-A9 protein.4. Intracellular detection of MAGE-A9 gene product was attempted in hepatocellular carcinoma tissues and a cytoplasmic reaction was obtained with anti-MAGE-A9 monoclonal antibody. Tissues of 39 hepatocellular carc更多还原