基于TEM-8新型颗粒性疫苗的构建、鉴定及其抗肿瘤免疫的研究

发布时间：2018-03-27 08:02

本文选题：肿瘤　切入点：TEM-8　出处：《第三军医大学》2010年硕士论文

【摘要】： 背景和目的: 实体瘤的生长和转移依赖于血管的生成,因此,抗肿瘤血管生成也成为抗肿瘤治疗的一种有效手段。目前,针对于VEGF为靶点的被动免疫抗血管生成的药物在临床上取得了一定进展后,随之而来的抗肿瘤血管生成治疗也在不断的深入,以激活细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)为主的主动免疫治疗策略在恶性肿瘤的综合治疗中发挥了重要作用。与其它传统的治疗方法相比较,主动免疫治疗能诱导自身的肿瘤特异性免疫应答,不但可以识别和清除肿瘤细胞,而且还可以诱发免疫记忆,阻止肿瘤的复发。同时能够减少持续给药给患者带来的麻烦和严重的经济负担。所以,抗肿瘤血管生成疫苗的运用前景非常具有吸引力。到目前为止,抗肿瘤血管生成的分子疫苗治疗中,靶抗原主要是VEGFR-2,VEGF,FGFR,MMP-2分子等,但是这些分子不仅在肿瘤细胞组织中表达丰富,而且在一些正常组织内表达量也较高,所以针对类似靶点设计的分子疫苗对自身有一定的潜在危害性。 2000年Croix发现了9条在结肠癌血管内皮中特异性高表达的基因,分别命名为肿瘤内皮标志物1～9(tumor endothelial markers,TEMs)。经原位杂交检测发现,在成年小鼠体内,TEM-8的mRNA在正常组织中不表达或者微量表达,而在其肿瘤内皮中的表达却非常丰富。在人体内,TEM-8mRNA和TEM-8蛋白几乎在所有的结肠癌、食管癌、膀胱癌、肺癌病例中高表达,而在同一病例的癌旁组织和正常组织中却不表达,在黄体及伤口愈合过程中的新血管中也没有检测出TEM-8蛋白分子。这些研究结果提示TEM-8是目前最理想的免疫治疗候选靶点之一。在后来的分子疫苗研究中证实TEM-8所构成的单独的分子疫苗免疫原性较低,而与其他抗原结合以后可以有效的激发免疫反应,后来的学者在临床上引入DC疫苗后可以有效的提高TEM-8的免疫原性,抗瘤效果明显,但是存在转运效率不高,操作不方便,费用高等特点。阳离子肽类DNA转运载体(cationic peptides DNA delivery systems)是进展较快的领域之一。该类DNA转运载体为一些富含正电荷氨基酸的多肽,如多聚赖氨酸(ploylysine,[K]n),它们可通过电性中和作用与富含负电荷(磷酸根基团)的质粒DNA发生聚合、压缩,从而将直径约数百纳米、松散的质粒DNA聚缩成为几十纳米的致密颗粒,使之易于被真核细胞摄入从而达到基因转染的目的。构建“肽-DNA复合疫苗”,这种疫苗可以打破自身耐受,操作方便,是比较理想的选择之一。本课题的目的就在于评估基于TEM-8的分子疫苗经过阳离子穿膜肽改造后的抗肿瘤免疫治疗的有效性和毒副作用,从而评价是否有必要对TEM-8进行更多的免疫治疗方面的研究。方法: 1.我们首先构建阳离子融合肽[K]16-tat49-57,融合肽经反相高压液相色谱鉴定其纯度,用质谱鉴定其分子量,在特定NaCl浓度下,将其与富含负电荷的pTEM-8真核表达质粒结合构建成新型颗粒性疫苗,用透射电镜扫描显示其颗粒大小,用westernblot检测TEM-8蛋白的表达; 2.构建CT-26结肠癌模型,我们以基于TEM-8的新型颗粒性疫苗免疫Balb/c小鼠,研究疫苗的抗瘤能力。免疫小鼠每周一次,连续三次。观察小鼠的生存时间和肿瘤体积变化情况,用免疫组化观察肿瘤组织血管生长情况,并计算肿瘤微血管密度; 3.确认了疫苗的抗肿瘤作用之后,我们进一步研究了疫苗的抗肿瘤机理和毒副作用; 结果: 1.对合成的多肽进行高效液相色谱鉴定其纯度达到了96.57%用质谱分析其分子量为3390.44 Da,并成功构建了颗粒性疫苗,透射电镜及软件分析,其颗粒大小均匀,直径小于25nm,符合疫苗要求,并对其转染和免疫印迹检测分子量为63KD符合预期结果; 2.实验结果显示,基于TEM-8的颗粒性疫苗免疫组的肿瘤体积与对照组有明显差异(p0.05),荷瘤小鼠生存期延长。肿瘤组织内血管内皮细胞采用抗CD31单克隆抗体作免疫组织化学染色,结果显示TEM-8疫苗免疫组微血管密度(MVD)较对照组明显降低; 3.在激发CTL的51Cr释放实验证明,经过疫苗免疫的小鼠能够激发CTL免疫反应,ELISPOT检测显示在体内疫苗能有效激发抗原特异性CTL效应,分别利用抗CD4+或者抗CD8+单抗作保护性免疫试验,结果发现抗CD8+时,小鼠不能获得疫苗免疫后的保护性效果;而在抗CD4+时,实验组和对照组小鼠肿瘤体积仍然有显著性差异(p0.05),提示CD8+T细胞在抗肿瘤中起主要作用; 结论: 1.成功构建基于TEM-8的新型颗粒性疫苗,同时能够有效介导基因的表达; 2.基于TEM-8的颗粒性疫苗能有效抑制肿瘤的生长速度、提高荷瘤动物的生存率; 3.CD8+T细胞在以DNA疫苗所诱导的抗肿瘤免疫应答中起主要作用;
[Abstract]:Background and purpose:
Generation, growth and metastasis of solid tumors depends on angiogenesis therefore, anti angiogenesis has become an effective means of anti-tumor therapy. At present, the drug for passive immunization of VEGF as a target of anti angiogenesis has made some progress in clinical, anti angiogenesis therapy has also been followed further, in order to activate cytotoxic T lymphocytes (cytotoxic T, lymphocyte, CTL) plays an important role in the comprehensive treatment of active immunotherapy based on malignant tumor. Compared with other traditional methods of treatment, active immunotherapy can induce tumor specific immune response itself, not only can identify and eliminate tumor cells. But also can induce immune memory, prevent the recurrence of the tumor. At the same time can reduce the continuous administration of patients brought trouble and serious economic burden. Therefore, anti tumor angiogenesis A vaccine application is very attractive. So far, the molecular vaccine anti angiogenic therapy, target antigen is mainly VEGFR-2, VEGF, FGFR, MMP-2 molecules, but these molecules not only in tumor tissue was abundant, and its expression level was also higher in some normal tissues, so the molecular vaccine similar target design has the potential danger to themselves.
2000 Croix found 9 high specific expression in vascular endothelial cells of colon cancer gene, named as tumor endothelial marker 1~9 (tumor endothelial markers, TEMs). By in situ hybridization detected in adult mice, TEM-8 mRNA expression or trace expression in normal tissues, and its expression in tumor in the endothelium is very abundant in the human body, TEM-8mRNA and TEM-8 protein in almost all colon cancer, esophageal cancer, bladder cancer, high expression of lung cancer cases, and in the same case paracancerous tissues and normal tissues but not expression in the corpus luteum and wound healing in the process of new blood vessels in no detection of TEM-8 protein. These results suggest that TEM-8 is one of the most ideal current immunotherapy candidate targets. Confirmed by TEM-8 single molecular vaccine immunogenicity low molecular vaccine in the later study, and After binding with other antigens, it can effectively stimulate the immune response. Later scholars introduced DC vaccine in clinic, which can effectively improve the immunogenicity of TEM-8, and has obvious anti-tumor effect. However, there are some characteristics such as low transport efficiency, inconvenient operation and high cost.
Cationic peptides DNA transporter (cationic peptides DNA delivery systems) is one of the rapid progress of the field. The class DNA transporter for some rich in positively charged amino acid polypeptide, such as polylysine (ploylysine, [K]n), which can be through the electrical neutralization and rich negative charge (phosphate). DNA polymerization, compression, which will be about hundreds of nanometer diameter, plasmid DNA loose condensation become dense particles of tens of nanometers, which is easy to eukaryotic cells so as to achieve the purpose of intake of gene transfection. Construction of peptide -DNA complex vaccine, this vaccine can break self tolerance, convenient operation, is one of the choice the ideal.
The purpose of this project is to evaluate the effectiveness and side effects of TEM-8 based molecular vaccine for anti-tumor immunotherapy after cationic transmembrane peptide modification, so as to evaluate whether it is necessary to carry out more immunotherapy for TEM-8.
Method:
We constructed 1. cationic fusion peptide [K]16-tat49-57 fusion peptide by reversed phase high pressure liquid chromatography. The purity and identification of the molecular weight by mass spectrometry, in particular NaCl concentration, with the rich negative charge pTEM-8 eukaryotic expression plasmid constructed with a novel particle vaccine, using transmission electron microscope scanning showed that the particle size, use to detect the expression of TEM-8 protein westernblot;
2. to construct the CT-26 model of colon cancer, we use a novel particle vaccine Balb/c mice were immunized with TEM-8 based vaccine antitumor ability of immunized mice. Once a week, three times in a row. The survival time and tumor volume changes were observed in mice, using immunohistochemical observation of tumor angiogenesis, and tumor microvessel calculation density;
3. after confirming the antitumor effect of the vaccine, we further studied the antitumor mechanism and side effects of the vaccine.
Result:
1. of the synthesized peptide by HPLC. The purity reached 96.57% by mass spectrometry analysis of the molecular weight of 3390.44 Da, and successfully constructed the particle vaccine, TEM analysis and software, the uniform particle size, diameter less than 25nm, meet the requirements of molecular detection and vaccine, the transfection and immunoblotting was 63KD in accordance with the expected results;
2.. Experimental results show that the TEM-8 based particle vaccine immune group of tumor size and the control group had significant difference (P0.05), prolong the survival time of tumor bearing mice. Vascular endothelial cells in tumor tissue using anti CD31 monoclonal antibodies for immunohistochemical staining in the immunized group TEM-8 vaccine microvessel density (MVD) decreased significantly compared with the control group;
3. excitation at CTL 51Cr release experiments show that after the vaccine can stimulate the immune response of CTL mice, ELISPOT assay can effectively stimulate antigen-specific CTL effect in vivo vaccine, respectively using anti CD4+ or anti CD8+ monoclonal antibody for protective immunity test results anti CD8+, protective effect in mice can not get vaccinated after; and in anti CD4+, the experimental group and the control group the tumor volume still has significant difference (P0.05), suggesting that CD8+T cells play a major role in anti tumor;
Conclusion:
1. the new granular vaccine based on TEM-8 was successfully constructed, and the gene expression could be effectively mediated.
2. TEM-8 based granular vaccine can effectively inhibit the growth rate of tumor and improve the survival rate of tumor bearing animals.
3.CD8+T cells play a major role in the anti-tumor immune response induced by DNA vaccine.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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