【作者】 阮志华；

【导师】 吴玉章；

【作者基本信息】 第三军医大学 ， 免疫学， 2008， 博士

【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(p<0.05),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(p<0.05),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。更多还原

【Abstract】 Angiogenesis is important for some normal physiologic progression such as embryonic development, wound healing, inflammation, but also important for some pathologic conditions such as tumor, rheumatic arthritis, retinopathies, etc. Direct and indirect evidences indicated that the growth and metastasis of tumor are angiogenesis-dependent. Therefore, tumor angiogenesis research has become a hot spot recently. Up to date, some specific anti-angiogenic drugs have been enrolled clinical trials and proved that anti-angiogenesis was a potential method for cancer therapy. Since anti-angiogenic therapy needs a persistent and long-term impact to suppress tumor growth, specific chemical or biological inhibitors of angiogenesis often require constant administration at a relatively high dose level that is very troublesome and costly. Compared with chemical therapy, active immunotherapy is more convenient, cost effective and worthy of intensive exploration. Recently, researchers have obtained some evidences that active immunotherapy can inhibit tumor growth in vivo, but some disadvantages such as the low specificity of the targeted antigen need to be improved.Tumor endothelial markers (TEMs) which dominantly expressed in endothelium of human colon cancer were observed by Croix and his colleagues in 2000. Following evidences indicated that TEM8 is one of the most specific antigens for anti-angiogenic tumor immunotherapy. However, whether TEM8 can act as an ideal target for anti-angiogenic therapy remains unclear. In this report, we tested our hypothesis that active immunotherapy targeting TEM8 could inhibit tumor angiogenesis and tumor growth.we firstly constructed the eukaryotic expression plasmid pTEM8-Ⅰencoding TEM8-Ⅰand bicistronic expression plasmid pTEM8/mGM-CSF containing human TEM8-Ⅰand murine GM-CSF. After identified with restriction enzymes and sequencing, the two plasmids were used to transform an attenuated strain of murine Salmonella typhimurium (SL7207) to obtain oral vaccines based on TEM8. At the same time, eukaryotic expression plasmid pEGFP-N1 encoding green fluorescent protein (GFP) was used to transform SL7207 to obtain a tracing vaccine. Then, C57BL/6 mice were immunized with these oral vaccines. 3 days later, antigen presentation cells expressing GFP were found both in Peyer’s patches and spleen, confirming that the vaccine was effectively processed and the immunization condition was optimal.We adopted B16F10 murine melanoma model and CT26 murine colon carcinoma model to estimate the vaccine’s anti-tumor efficacy by protective setting or therapeutic setting. In protective experiment, mice were immunized three times at 2-wk intervals. Two week thereafter, mice were challenged with tumor cells by s.c. or i.v. injection to induce primary tumor or experimental pulmonary metastases. In therapeutic experiment, mice were challenged with tumor cells 3 days after the first immunization, then the other two times vaccinations were performed at 2-wk intervals as before. We found that immunotherapy with SL7207/pTEM8-Ⅰor SL727/pTEM8/mGM-CSF primed effective anti-tumor immune response at both protective and therapeutic settings, resulting in markedly reduced growth of tumors and prolonged life span of immunized mice.We also used a monoclonal antibody specific for murine CD31 to detect the microvessel density of tumor tissues, and observed that MVD of tumors in mice immunized with TEM8 vaccines is obviously lower than the control group. The results of alginate assay in vivo and FITC-Dextran ingestion assay also indicated that angiogenesis in immunized mice was inhibited. To detect TEM8 specific CTLs, splenocytes were collected from mice immunized with pTEM8/mGM-CSF or control group, and tested by a standard 51Cr release assay. The results showed that only splenocytes isolated from the mice immunized with pTEM8/mGM-CSF killed the CT26 cells transfected with TEM8 gene. A multiclonal antibody specific for TEM8 was used to detect the expression profile of TEM8 in CT26 colon carcinoma tissues, the results showed that the TEM8 proteins only expressed in tumor endothelial cells. These results further demonstrated that the anti-tumor activity of TEM8 vaccines is resulted from destroying the tumor endothelial cells.At last, we explored the vaccine’s anti-tumor mechanism and side effects. We did the experiment of protective immunization utilizing the C57BL/6J derived CD4 gene knockout mice and CD8 gene knockout mice. No obvious protective immune response was observed in mouse immunized with the vaccine in the absence of CD8 molecules; but the CD4 deficiency seemed do not affect the vaccine induced protective immune response. These results implied that CD8+ T cells play the essential role in the anti-tumor immune response primed by the vaccines. As to side effects, we didn’t find obvious differences between the mice orally immunized with pTEM8/mGM-CSF with control group in body weight, major viscera appearance and general state. The skin vulneration assay in the two groups also showed no difference in skin wound healing and histologic characteristics.Based on the results above, we can draw following conclusions:①Oral DNA vaccines delivered by attenuated salmonella have the ability to break self-tolerance and induce the immune response against self antigens;②The vaccines targeting the TEM8 can inhibit tumor growth by anti-angiogenesis effects;③CD8+ T cells play the critical role in tumor rejection immune response primed by oral DNA vaccine delivered by attenuated salmonella.更多还原