基于TEM8的口服疫苗构建及其抗肿瘤作用研究

发布时间：2019-06-14 22:54

【摘要】： 血管生成是胚胎发育、伤口愈合、炎症中的生理反应,但是在实体瘤、风湿性关节炎、糖尿病性视网膜炎等疾病中却属于病理过程。众多的证据表明,实体瘤的生长和转移依赖于血管生成,肿瘤血管生成也因此成为肿瘤学研究的热点领域之一。目前,部分化学合成的抗血管生成的药物已进入临床试验,并证明抗血管生成是有效的肿瘤治疗方法。由于抗血管生成是一个长期的治疗过程,持续给药不仅麻烦而且给患者带来很重的经济负担。主动免疫治疗在少数几次给药的情况下,便可以获得长期的保护性效果,不但方便而且便宜,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,主动免疫治疗在动物实验中取得了一定的疗效,但还存在一些缺点,比如靶抗原的特异性不高等等。2000年Croix发现多条在结肠癌血管内皮中特异性高表达的基因,命名为肿瘤内皮标志物(tumor endothelial markers,TEMs)。随后的研究提示提示TEM8是目前特异性最高的免疫治疗候选靶抗原,但TEM8是否为抗肿瘤治疗的有效靶抗原并没有得到证明。本课题的目的就在于评估基于TEM8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TEM8进行更多的免疫治疗方面的研究。我们首先构建编码人TEM8-Ⅰ基因的真核表达质粒pTEM8-Ⅰ和编码人TEM8-Ⅰ、小鼠GM-CSF基因的双表达质粒pTEM8/mGM-CSF。经限制性酶切和测序鉴定后转化营养缺陷型小鼠沙门氏菌株SL7207,制备基于TEM8的口服疫苗。以编码绿色荧光蛋白的真核表达质粒pEGFP-N1转化SL7207后制备示踪疫苗,口服免疫C57BL/6小鼠,3天后,在Peyer’s结和脾细胞中均可发现表达绿色荧光蛋白的抗原提呈细胞,确认了这种疫苗传递抗原的有效性和最佳条件。随后,我们以pTEM8-Ⅰ和pTEM8/mGM-CSF免疫C57BL/6J小鼠,取脾,以羊抗小鼠TEM8特异性抗体作免疫组化,检测到TEM8蛋白在脾内APC的细胞膜和细胞浆中表达,证实了所构建的疫苗能够完成质粒DNA由细菌到体内APC的传递,以及在APC内表达目的抗原的工作。在证实上述载体的抗原传递能力之后,我们研究了疫苗的抗肿瘤能力。主要包括保护性免疫和治疗性免疫两种方案,在B16黑色素瘤和CT26结肠癌模型中完成。在保护性免疫组,给小鼠口服免疫3次后,皮下接种肿瘤细胞建立皮下肿瘤模型或者经尾静脉注射肿瘤细胞建立肺转移瘤模型;在治疗性免疫组,在第一次口服免疫后3天接种肿瘤细胞,按免疫方案继续给小鼠口服免疫2次。实验结果显示,以TEM8为靶抗原的抗肿瘤血管生成疫苗,对这两种肿瘤都有明显的抑制作用,其中以CT26结肠癌皮下模型最明显;TEM8-Ⅰ和pTEM8/mGM-CSF免疫组的肿瘤体积、肺转移瘤的数目与对照组有明显差异(p0.05),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TEM8疫苗免疫组微血管密度(MVD)较对照组明显降低;藻酸盐体内实验显示TEM8疫苗免疫组肿瘤血管生成明显少于对照组,FITC-Dextran摄取率也低于对照组。以pTEM8/mGM-CSF免疫后,取脾细胞以标准51Cr释放试验检测TEM8特异性CTL,显示免疫组小鼠脾细胞对CT26无杀伤作用,但可以杀伤对转染了TEM8基因的CT26细胞。以羊抗鼠TEM8抗体A16作免疫组织化学检测证实CT26结肠癌组织中只有血管内皮细胞表达TEM8蛋白,进一步说明TEM8疫苗免疫后,是通过杀伤肿瘤血管内皮细胞取得抗肿瘤效果。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。分别利用CD4或者CD8基因敲除小鼠作保护性免疫试验,结果发现CD8缺陷时,小鼠不能获得疫苗免疫后的保护性效果;而在CD4缺陷时,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细胞在抗肿瘤中取主要作用。给小鼠口服免疫pTEM8/mGM-CSF后观察体重、主要内脏、一般情况等,未发现明显差别。皮肤致伤实验结果显示pTEM8/mGM-CSF免疫小鼠和对照组皮肤伤口的愈合时间和组织学特征均无显著差别,提示该疫苗免疫后无明显副作用。综合以上研究,可以得出以下结论:①以减毒沙门氏菌SL7207为载体的口服DNA疫苗可以打破自身耐受,诱导针对自身抗原的免疫反应;②以TEM8为靶抗原的疫苗可通过抗血管生成发挥抗肿瘤作用;③CD8+T细胞在以减毒沙门氏菌为载体的口服DNA疫苗所诱导的抗肿瘤免疫应答中取主要作用。
[Abstract]:Angiogenesis is a physiological response in embryonic development, wound healing and inflammation, but it is a pathological process in solid tumors, rheumatic arthritis, and diabetic retinitis. Numerous evidence suggests that growth and metastasis of solid tumors depend on angiogenesis and tumor angiogenesis is also one of the hot spots in oncology research. At present, some of the chemically synthesized anti-angiogenic drugs have entered clinical trials and demonstrate that anti-angiogenic is an effective method of tumor therapy. Because anti-angiogenesis is a long-term treatment process, continuous administration is not only a problem but also a heavy economic burden for patients. Active immune therapy can obtain a long-term protective effect in a few times of administration, and is not only convenient and cheap, so the application prospect of the anti-tumor angiogenesis vaccine is very attractive. So far, active immunotherapy has achieved a certain therapeutic effect in animal experiments, but there are some disadvantages, such as the high specificity of the target antigen and so on. TEMs). The subsequent study suggests that TEM8 is the most specific candidate target antigen with the highest specificity, but whether TEM8 is the effective target antigen for anti-tumor therapy is not shown. The purpose of this study is to evaluate the effectiveness and toxic and side effects of TEM8-based anti-tumor immunotherapy, so as to evaluate the need for more immunotherapy for TEM8. We first construct the eukaryotic expression plasmid pTEM8-I of the human TEM8-I gene and the double-expression plasmid pTEM8/ mGM-C of the human TEM8-I and the mouse GM-CSF gene. SF. transformed auxotrophic Salmonella strain SL7207 after restriction enzyme digestion and sequencing to prepare a TEM8-based oral administration The vaccine was prepared by transforming SL7207 with the eukaryotic expression plasmid pEGFP-N1 of the green fluorescent protein. After 3 days, the antigen-presenting cells expressing the green fluorescent protein can be found in the Peyer's junction and the spleen cell, and the effectiveness and the optimum of the vaccine delivery antigen are confirmed. Conditions. Subsequently, we immunized C57BL/ 6J mice with pTEM8-I and pTEM8/ mGM-CSF, and the spleen and the mouse TEM8 specific antibody were used as immunohistochemistry to detect the expression of the TEM8 protein in the cell membrane and the cytoplasm of the APC in the spleen, and it was confirmed that the constructed vaccine was able to complete the plasmid DNA from the bacteria to the in vivo APC. And the expression of the target antigen in the APC. Work. After confirming the antigen delivery ability of the above-mentioned vector, we studied the vaccine. Anti-tumor ability. It mainly includes both protective and therapeutic immunization protocols, in B16 melanoma and CT26 colon cancer The model was completed. In the protective immune group, after three times of oral immunization of the mice, the tumor cells were inoculated subcutaneously to establish a subcutaneous tumor model or a lung metastasis tumor model was established by the tail vein injection of the tumor cells; in the therapeutic immunization group,3 days after the first oral immunization, Tumor cells, continued to the mouse port according to the immune protocol The results of the experiment show that the anti-tumor angiogenesis vaccine with TEM8 as the target antigen has an obvious inhibitory effect on the two tumors, in which the tumor volume of the CT26 colon cancer subdermal model is the most obvious; the number of the lung metastases in the TEM8-I and pTEM8/ mGM-CSF immune groups is significantly different from that of the control group (p 0.05), mouse The results showed that the microvessel density (MVD) in the immune group of the TEM8 vaccine was significantly lower than that in the control group. In less than the control group, the FITC-Dextran uptake rate was also After immunization with pTEM8/ mGM-CSF, the spleen cells were taken to test the TEM8-specific CTL with the standard 51Cr release test. T26 cells. The expression of TEM8 protein in the vascular endothelial cells in the CT26 colon cancer tissue was confirmed by the immunohistochemical study of the goat anti-mouse TEM8 antibody A16, and it was further demonstrated that the TEM8 vaccine was immunized with the anti-tumor vascular endothelial cells. The anti-tumor effect of the vaccine is confirmed, and the anti-swelling of the vaccine is further studied after the anti-tumor effect of the vaccine is confirmed. The mechanism of the tumor and the toxic and side effects of the mice were tested by using the CD4 or CD8 gene knockout mice. The results showed that the mice were unable to obtain the protective effect after the vaccine immunization, and the volume of the tumor in the experimental group and the control group was still significant in the case of the CD4 defect. Heteropoly (p0.05), suggesting that CD8 + T cells are resistant to The main role of the tumor is to observe body weight, main internal organs, general conditions, etc. after oral administration of pTEM8/ mGM-CSF to mice. No significant difference was found. The results of skin injury showed no significant difference in the healing time and the histological characteristics of the skin wound in the mice and the control group. The following conclusions can be obtained by combining the above studies: the oral DNA vaccine which is the carrier of the attenuated Salmonella SL7207 can break the self-tolerance and induce the immune response to the autoantigen; and the vaccine with the TEM8 as the target antigen can be used as a target antigen. Anti-tumor effect of blood vessel production; anti-swelling induced by oral DNA vaccine with attenuated Salmonella as carrie
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392;R730.5

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