【作者】 黄亚渝；

【导师】 隋延仿；

【作者基本信息】 第四军医大学 ， 病理学与病理生理学， 2005， 博士

【摘要】 长久以来,恶性肿瘤一直严重威胁着人类的健康和生命,传统的治疗方法不尽如人意。免疫治疗通过提高肿瘤的免疫原性,诱导机体产生特异性的抗肿瘤免疫反应,达到治疗肿瘤的目的,是一种变被动为主动的生物治疗方法。在研究者的共同努力下,有关肿瘤免疫治疗的基础和临床应用研究已取得一定的进展,在肿瘤的防治上也显示出一定的效果。但是人们仍在不断地探索和尝试,力图研制出高效、广谱的肿瘤疫苗,以挽救更多肿瘤患者的生命,给人们带来新的希望。 MAGE-n是由我室首次报道的,克隆自肝细胞癌细胞系的,MAGE家族基因的新成员(GeneBank登录号AF443295),它与MAGE-A亚家族基因有较高的同源性。MAGE-n分子上存在可诱导MAGE-n特异性CTL的抗原表位,而且在体内能产生特异性的抗肝癌免疫效应。CD80是一种共刺激分子,在T细胞的活化和抗肿瘤免疫反应中起着重要作用。CpG作为一种免疫佐剂,可以诱发机体产生多种免疫学效应。本研究构建MAGE-n与CD80融合基因肿瘤疫苗,以CpG为佐剂,研究其对小鼠细胞免疫及体液免疫的影响,并利用小鼠肿瘤模型观察对肿瘤的防治作用。 1.MAGE-n基因的克隆、原核表达、抗血清的制备及其在不同组织中的表达更多还原

【Abstract】 Tumor is always a big threat against people’s health and life, and the available protocols are far from satisfaction. Tumor biological therapies, which can elicit antigen-specific antitumor immunity and play an important role in prevention and therapy of tumor, are regarded as the most attractive method. Scientists have made great progresss in basal and clinical research of tumor biological therapies. However people are still exploring and trying to find a more efficient, more broad-spectrum tumor vaccine to save more patients’ life, which will bring a hope for humankind.MAGE-n is a new member of MAGE gene family, which was firstly reported by our research group (GeneBank, Locus No.AF443295), and cloned from Hepatocellular carcinoma cell line (HCCC). It is highly homologous to MAGE-A subfamily genes. The CTL epitopes of MAGE-n were identified and proved to elicit MAGE-n-specific CTLs, and to be a potential target of specific immunotherapy for HLA-A2+ HCC patients in the future. CD80 is one of costimulating factor, which plays a key role in T cells activation and antitumor immunity. As a kind of adjuvant, CpG-ODN can induce great immunological effects. In our research, the MAGE-n and CD80 fusion DNA vaccine was constructed. Conjucted with CpG-ODN adjuvant, the humoral- and cellular-activating effects of the fusion DNA vaccine were measured, and the therapeutic and protective effects of the vaccines against established tumor expressing MAGE-n were investigated.1: Cloning, Prokaryotic Expression of Human MAGE-n, Preparation of Antiserum, and Expression of MAGE-n in Different TissuesObjective: To construct MAGE-n prokaryotic expression plasmid. To express the MAGE-n gene in E. coli, and to purify the protein. To obtain the anti-MAGE-n polyclonal antibody. To investigate the expression of MAGE-n in different tissues. Methods: The full-length ORF of MAEG-n gene was amplified by PCR from HHCC. Antigenicity of MAGE-n was analyzed by biological software. 300 bp segment of MAGE-n gene was amplified by PCR. The expression plasmid pGEX-MAGE-n was constructed, the DH5a containing the plasmid was induced by IPTG and the protein was purified with GSTrap FF column. Antiserum against MAGE-n was prepared by immunizing rabbit with the purified recombinant MAGE-n protein conjucted with Freund’s adjuvant. The anti-MAGE-n antiserum was purified through (NH4)2SO4 precipitation and CNBr-Activated Sephrose 4B chromatography, which was identified by agar gel precipitation, ELISA, and Western blot. The immunohisto-chemical analysis was used to detected the expression of MAGE-n in tumor, infiammantion and normal tissues. Results: The MAGE-n gene was cloned from HHCC, and the sequence was identical with that reported in GeneBank. The 100 aa (88-187 aa) segment of MAGE-n had a high antigenicity and specificity. The pGEX-MAGE-n plasmid was successfully constructed and the DH5a containing the plasmid could express a 40 kD protein whose purity was 82%. High titer antiserum against MAGE-n was obtained in immunized rabbits and the specificity to MAGE-n was increased by affinity chromatography. Immunocytochemical analysis with this antibody showed that MAGE-n expressed in cytoplasm. MAGE-n expressed in malignant tumor tissues and HHCC, 7402 cell lines, and not expressed in benign tumor, infiammantion, normal tissues and HepG2, 9204, 7721 cell lines. Conclusion: The protein of human MAGE-n segment was expressed and purified firstly. A high titer, specific rabbit antiserum against MAGE-n was prepared successfully. The expression of MAGE-n in different tissues was measured by the method of immunohistochemistry, which was identical with that of MAGE-A genes. The study prepared the materials for the further research of the MAGE-n as a promising candidate for tumor vaccine.2: The Constructions and Expression of MAGE-n and CD80 Fusion PlasmidObjective: To construct CD80, MAGE-n and MAGE-n/CD80 fusion plasmid and investigate their expression in vivo and vitro. To establish the B16 cell stably expressing MAGE-n. Methods: The eukaryotic expression plasmids pcDNA3.1+-CD80 and pcDNA3.1+-CD80/IRES/MAGE-n were constructed, and the later was transfected to NIH3T3 through Lipofectamine? 2000. By the method of FCM and immunocytochemistry, the expression of MAGE-n and CD80 were detected in transfected NIH3T3. Recombinant plasmid and Lipofectamine were injected on the leg of C57BL/6 mouse. Through semi-quantification RT-PCR and immunohistochemistry, MAGE-n and CD80 were proved to express in muscular tissues. The pcDNA3.1+-MAGE-n plasmid was constructed, then transfected into the mouse melanoma B16 cells under mediation of Lipofectamine. The positive clones were selected by G418. MAGE-n DNA, mRNA and protein in positive clones were detected by PCR, RT-PCR, Western blot and immunocytochemistry respectively. Results: pcDNA3.1+-CD80 and pcDNA3.1+-CD80/IRES/MAGE-n plasmids were constructed successfully. After transfection, CD80 was tested to express on the membrane of NIH3T3, and MAGE-n express in the plasm of NIH3T3. The MAGE-n positive materials were detected in the plasm of transfected NIH3T3. In the muscular tissues injected with the recombinant plasmid, MAGE-n and CD80 mRNA were detected by RT-PCR. In the plasm of muscular cells MAGE-n protein was detected by immunohistochemistry. The pcDNA3.1+- MAGE-n plasmid was constructed and transfected into B16 cells successfully. MAGE-n DNA, mRNA and protein in positive clones were detected respecttively. Conclusion: The coexpression plasmid pcDNA3.1+-CD80/IRES/MAGE-n was constructed successfully. Both MAGE-n and CD80 were tested to express in vivo and vitro. The B16-MAGE-n cell line stably expressing MAGE-n was obtained. These results lay a foundation for the research on antitumor effects of MAGE-n/CD80 fusion DNA vaccine.3: The Antitumor Effects of MAGE-n/CD80 Fusion DNA VaccineObjective: To evaluate the immunological effects in vivo, and protective and therapeutic effects of MAGE-n/CD80 fusion DNA vaccine on tumor.更多还原