【作者】 石永玉；

【导师】 陈慰峰； 孙汶生；

【作者基本信息】 山东大学 ， 免疫学， 2004， 博士

【摘要】 肿瘤抗原及其编码基因的研究不仅促进了肿瘤免疫学的理论发展,而且开辟了以肿瘤抗原为靶位的肿瘤特异性免疫治疗的新纪元。目前,肿瘤抗原可分为以下几类:(1)组织特异性分化抗原,(2)基因突变所致的抗原,(3)过量表达的抗原,(4)病毒编码的抗原,(5)不同剪切造成的变异体抗原,(6)CT抗原(cancer-testis antigen)。其中CT抗原被认为是肿瘤特异性的抗原,因为这一类抗原在多种肿瘤组织中表达,而在睾丸、卵巢和胎盘除外的正常组织中无表达,所以也是最有希望应用于免疫治疗的一类抗原。现在试用于临床上的主要就是这一类抗原,如NY-ES0-1。肿瘤免疫治疗的前提和关键是获得特异性的肿瘤抗原,鉴定肿瘤抗原的方法有多种:CTL筛选法、抑制消减杂交技术(suppression subtractive hybridization, SSH)、重组cDNA表达文库的血清学分析法(serological analysis of recombinant cDNA expression libraries, SEREX)、多肽洗脱法以及利用基因信息学的数据库开采法等。其中的SEREX方法因其操作简单、结果可靠而得以广泛应用。近年来,应用SEREX方法发现了多种肿瘤抗原,这些肿瘤抗原及其编码基因的鉴定,不仅为肿瘤的诊断、治疗及预后分析提供了靶位与分子标志,并为探讨肿瘤发生、发展的分子机理提供了依据。 目前,肿瘤常规治疗方法很难达到治愈目的,肿瘤特异性免疫有望成为肿更多还原

【Abstract】 It is a new era for the study of tumor antigens and tumor immunology when human tumor antigens and their encoding genes are identified. Subsequently, the tumor specific immunotherapy is making progress. At present, the majority of tumor antigens fall into one or more of the following categories: (1) differentiation antigens, (2) mutational antigens, (3) overexpressed antigens, (4) viral antigens, (5) alternative splicing variant antigens, (6) cancer - testis (CT) antigens. The CT antigens are considered as tumor specific antigens because they are expressed in a wide range of different histological types of cancer but generally not in other normal tissues except testis, ovary or placenta. Therefore, CT antigens are the first candidates for tumor immunotherapy and are used in clinic trial, such as NY-ESO-1. Since the identification of tumor antigens is the precondition for clinic immunotherapy, many approaches have been developed to identify tumor antigens, such as CTL cloning method, suppression subtractive hybridization (SSH)assay, serological analysis of recombinant cDNAexpression libraries(SEREX), peptide-eluting method and database-mining method. SEREX method is based on humoral immune reactivity to cancer. During the recent several years, a number of tumor antigens have been found by SEREX. The identification of these tumor antigens provides candidates for immunological applications and molecular features for understanding tumorigenesis.At present, it is difficult for traditional therapy to cure malignant diseases. Immunotherapy is limited by some factors though it brings a promise from preventing recurrence post-surgical. The immunotherapy targetting one kind of tumor antigen is not effective because the tumor antigen is heterogeneously expressed in tumor tissues and it could disappear to escape from the immne response. It is an important strategy that several tumor antigens are combined and used as vaccines in immunotherapy. Identification of more tumor antigens is required for effective immunotherapy. To identify tumor antigens expressed in HCC cells further, we constructed a HCC cDNA expression library and screened it with autologous sera of the patients. Moreover, the mRNA expression and serological reaction were analyzed in HCC, gastric cancers and colorectal cancers. Our results provided candidate targets for tumor immunotherapy and helped to clarify mechanisms of tumorigenesis.Research work described in this thesis consists of two parts. The first part is about establishing a HCC cDNA expression library and identifying genes of tumor associated antigens. The second part is about CAGE antigen which was analyzed for its mRNA expression and serological reactivity in HCC, gastric cancers and colorectal cancers. PART ONEObjective: The aim of this part is to identify tumor antigens in human hepatocellular carcinoma in order to provide targets for tumorimmunotherapy and clues for understanding tumorigenesis. Method: HCC associated antigens were identified by SEREX method.1. Construction of cDNA expression library. Total RNAs were extracted from two HCC tissues, then their mRNAs were purified and mixed at equal amounts. The cDNA was synthesized by reverse transcription. EcoR I adapters were ligated after blunting the cDNA termini. Then the cDNA was digested with Xho I and purified by SizeSep400 Spun Column. The product of purification was quantitated by ethidiura bromid plate assay, then was ligated into the ZAP express vector. After in vitro packaging, a primary phage expression library was obtained. The primary library was titered and part of it was amplified.2. Immunological screening of the cDNA expression library. The host bacteria infected with phages from the primary library were plated onto agar plates with isopropylthio-B-D-galactoside (IPTG). Phage plaques appeared after incubation and the expressed proteins in the plaques were transfered into nitrocellulose membranes. The membranes were reacted with pooled sera obtained from two patients (preabsorbed with phage-infected Escherichia coli lysate). Reactive clones were visualized by coloring with 4-nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP). These clones were subcloned two times to obtain monoclones.3. Identification of cDNA sequences in positive clones and bioinformatic analysis. Positive phage clones were converted to pBK-CMV phagemids by in vivo excision. The phagemids were purified and the sizes of the cDNA inserts were determined by restriction enzyme digestion with EcoR I and Xho I. The cDNA inserts were sequenced and analyzed by BLAST in GeneBank database (http://www. ncbi. nlm. nih. gov).Results: The recombinant efficiency of primary library was 98%. The size of the primary library was 1. OX 10*pfu, and its titer was 2. OX 106pfu/ml.Fifty-two clones reacted with the pooled sera from the two HCC patients were isolated and the inserted cDNAs were sequenced. Through similarity search in GenBank database, thirty-one genes of hepatocellular carcinoma-associated tumor antigens are identified, of which one is unknown and thirty are known. The proteins encoded by these known genes were classified into seven categories: RNA transcription and splicing-associated molecules, protein metabolism-associated molecules, energy synthesis-associated molecules, signal transduction molecules, cell adhesion molecules, immunosuppressive molecules, and other proteins. Among these genes, CAGE, a cancer-test is (CT) antigen, was the first time to be found in HCC.Conclusion: It is the first time that CAGE, a cancer-testis (CT) antigen, was found in the HCC cDNA expression library. Identification of hepatocellular carcinoma-associated tumor antigens provides potential targets for immunotherapy of HCC patients and facilitates explanation of carcinogenesis of HCC. PART TWOObjective: The expression of CAGE gene and the humoral immune response naturally elicited by CAGE antigen were analyzed to demonstrate that CAGE antigen is a candidate target for immunotherapy in hepatocellular carcinomas, gastric cancers and colorectal cancers. Methods: The expression frequency of CAGE gene was detected by RT-PCR. Total RNA from cancer tissues and paired adjacent noncancerous tissues was extracted and treated with RNase-free DNase before reverse transcription with RT-for-PCR Kit. Gene-specific PCR primers were designed and synthesized to amplify cDNA fragments. The integrity and quantity of the cDNA were evaluated by the amplification of G3PDH. RT-PCR of CAGE was performed with 35 cycles of 30s at 94X:, 25s at 60V, and 30s at 72T:, followed by 7 min at 72"C. The reaction yielded a 529-bp更多还原