基于HPV16、18 E6E7基因肿瘤治疗性疫苗研究
发布时间：2018-07-18 16:16
【摘要】：宫颈癌是常见的妇科恶性肿瘤之一，占全世界妇女癌症死亡率第二位，仅次于乳腺癌。在某些发展中国家宫颈癌死亡率甚至居首位。宫颈癌的传统治疗方法有手术、放疗、化疗等，但这些手段各有其局限性。研究表明，HPV是宫颈癌的主要致病因子，几乎所有的宫颈癌发病都与高危型HPV感染有关。本课题以宫颈癌治疗性疫苗为中心，开展了两个方向的研究，即基于HPV16E6E7基因疫苗的抗肿瘤免疫反应研究以及优化突变融合基因HPV18E6E7的去致癌性检测。 E6和E7蛋白是高危型HPV的主要致癌蛋白，E6蛋白通过结合并降解P53蛋白，，破坏正常的细胞周期调控，促使细胞永生化；E7蛋白则通过结合并降解pRb蛋白而使其丧失抑癌功能。E6和E7蛋白在正常组织中不表达，只表达于HPV感染组织中，因此E6和E7蛋白是制备宫颈癌治疗性疫苗的理想靶抗原。 在疫苗的抗肿瘤免疫反应研究中，以优化突变融合的HPV16E6E7基因为抗原基因，构建重组腺病毒疫苗和重组DNA疫苗，另外用表达野生型和突变型E6E7蛋白的重组腺病毒感染小鼠树突状细胞，得到致敏树突状细胞疫苗。用ELISPOT检测疫苗免疫小鼠后诱导的细胞免疫反应；通过肿瘤移植保护实验检测疫苗的抗肿瘤效果。结果表明致敏树突细胞引发了强烈的细胞免疫反应，同时具有较好的抗肿瘤免疫效果。在优化突变融合基因HPV18E6E7的去致癌性检测中，本课题参照人类基因中的优势密码子，对HPV18E6基因和E7基因进行优化合成，同时对E6基因和E7基因进行定点突变和融合，以消除其致癌性。实验表明两种优化突变融合的HPV18E6E7基因失去了对NIH3T3细胞的转化活性和对BALB/c裸鼠的致瘤作用，表明优化融合突变基因是安全的。
[Abstract]:Cervical cancer is one of the most common gynecological malignancies, accounting for the second highest cancer mortality rate in the world, second only to breast cancer. In some developing countries, cervical cancer mortality even ranks first. The traditional treatment of cervical cancer includes surgery, radiotherapy, chemotherapy and so on, but each of these methods has its limitations. Studies have shown that HPV is the main cause of cervical cancer, almost all cervical cancer is associated with high-risk HPV infection. This paper focuses on the therapeutic vaccine for cervical cancer, and has carried out two research directions. That is, the study of anti-tumor immune response based on HPV16E6E7 gene vaccine and the optimized detection of carcinogenicity of mutant fusion gene HPV18E6E7. E6 and E7 proteins are the main carcinogenic proteins of high risk HPV. Disrupting normal cell cycle regulation, impelling cell immortalized E7 protein to lose its tumor suppressor function by binding and degrading PRB protein, E6 and E7 proteins were not expressed in normal tissues, but only in HPV infected tissues. Therefore, E6 and E7 proteins are ideal target antigens for the preparation of therapeutic vaccines for cervical cancer. The recombinant adenovirus vaccine and recombinant DNA vaccine were constructed by optimizing the fusion of HPV16E6E7 gene as antigen gene in the study of anti-tumor immune response of the vaccine. In addition, the mouse dendritic cells were infected with recombinant adenovirus expressing wild type and mutant E6E7 protein, and the sensitized dendritic cell vaccine was obtained. Elisa pot was used to detect the cellular immune response induced by the vaccine, and the anti-tumor effect of the vaccine was tested by tumor transplantation protection test. The results showed that the sensitized dendritic cells induced a strong cellular immune response and had a good antitumor immune effect. In the detection of the decarcinogenicity of the mutant fusion gene HPV18E6E7, HPV18E6 gene and E7 gene were optimized and synthesized according to the dominant codon of human gene, and the E6 gene and E7 gene were mutated and fused. To eliminate its carcinogenicity. The results showed that the two optimized fusion genes of HPV18E6E7 lost the transformation activity of NIH3T3 cells and the tumorigenicity of BALB- / c nude mice, which indicated that the optimized fusion gene was safe.
【学位授予单位】：北京工业大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33
本文编号：2132460
[Abstract]:Cervical cancer is one of the most common gynecological malignancies, accounting for the second highest cancer mortality rate in the world, second only to breast cancer. In some developing countries, cervical cancer mortality even ranks first. The traditional treatment of cervical cancer includes surgery, radiotherapy, chemotherapy and so on, but each of these methods has its limitations. Studies have shown that HPV is the main cause of cervical cancer, almost all cervical cancer is associated with high-risk HPV infection. This paper focuses on the therapeutic vaccine for cervical cancer, and has carried out two research directions. That is, the study of anti-tumor immune response based on HPV16E6E7 gene vaccine and the optimized detection of carcinogenicity of mutant fusion gene HPV18E6E7. E6 and E7 proteins are the main carcinogenic proteins of high risk HPV. Disrupting normal cell cycle regulation, impelling cell immortalized E7 protein to lose its tumor suppressor function by binding and degrading PRB protein, E6 and E7 proteins were not expressed in normal tissues, but only in HPV infected tissues. Therefore, E6 and E7 proteins are ideal target antigens for the preparation of therapeutic vaccines for cervical cancer. The recombinant adenovirus vaccine and recombinant DNA vaccine were constructed by optimizing the fusion of HPV16E6E7 gene as antigen gene in the study of anti-tumor immune response of the vaccine. In addition, the mouse dendritic cells were infected with recombinant adenovirus expressing wild type and mutant E6E7 protein, and the sensitized dendritic cell vaccine was obtained. Elisa pot was used to detect the cellular immune response induced by the vaccine, and the anti-tumor effect of the vaccine was tested by tumor transplantation protection test. The results showed that the sensitized dendritic cells induced a strong cellular immune response and had a good antitumor immune effect. In the detection of the decarcinogenicity of the mutant fusion gene HPV18E6E7, HPV18E6 gene and E7 gene were optimized and synthesized according to the dominant codon of human gene, and the E6 gene and E7 gene were mutated and fused. To eliminate its carcinogenicity. The results showed that the two optimized fusion genes of HPV18E6E7 lost the transformation activity of NIH3T3 cells and the tumorigenicity of BALB- / c nude mice, which indicated that the optimized fusion gene was safe.
【学位授予单位】：北京工业大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33
【引证文献】
相关期刊论文 前2条
1 张玉;孙立新;;人乳头瘤病毒E6/E7蛋白在子宫颈癌中的研究进展[J];肿瘤研究与临床;2016年08期
2 罗小兰;毛熙光;;HPV16E7蛋白在宫颈癌中的作用研究进展[J];现代医药卫生;2016年04期
相关硕士学位论文 前1条
1 张玉;HPV E6癌蛋白检测在宫颈癌筛查中的临床应用研究[D];山西医科大学;2016年
本文编号：2132460
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