随着手术和化疗的发展，结直肠癌（CRC）的治疗策略不断进步，但在疗效和不良副反应方面仍面临严峻的挑战。最近免疫治疗得到大家的广泛认可，对某些类型的癌症也包括结直肠癌的治疗取得了突破性的进展。同时，优于传统疫苗的新型疫苗正在研发。以重组抗原或多肽为基础设计的多肽疫苗由于具有良好的特异性和安全性，有望成为传统疫苗的替代品。从另一方面来说，聚合物纳米颗粒具有强大的负载抗原能力，较好的生物相容性，灵活的可修饰性等特点使其作为抗原载体在肿瘤免疫治疗中具有突出的优势。因此作为新型疫苗的纳米颗粒疫苗正在受到越来越多的关注。然而，有效地激活体内抗肿瘤免疫以达到治疗肿瘤的目的仍然是开发疫苗的一个重大的挑战。

本文采用生物相容性良好、负载量高的聚多巴胺纳米颗粒（PDA NPs）负载肿瘤模式抗原OVA或肿瘤细胞裂解物，并对其进行了体内外免疫学评估及抗肿瘤效果测试，评价PDA NPs负载单一抗原及复合抗原后对结肠癌荷瘤鼠抗肿瘤免疫效果，为PDA NPs应用到肿瘤免疫领域提供基础实验数据和参考。论文主要分为以下三个部分：

第一部分描述了携带肿瘤模式抗原OVA和绿色荧光蛋白（GFP）的OVA-GMC38结肠癌细胞系的构建。首先设计并构建了携带肿瘤模式抗原OVA和GFP报告基因的双基因慢病毒载体。采用嘌呤霉素在该慢病毒感染的MC38细胞中筛选阳性OVA-GMC38细胞。通过激光共聚焦显微镜、流式细胞术、蛋白质印迹（Western Blot）和定量逆转录聚合酶链反应（qRT-PCR）检测出GFP和OVA基因在MC38细胞中稳定表达。在C57BL/6小鼠皮下种植OVA-GMC38，考察其对荷瘤小鼠动物模型建立的影响，实验结果表明与MC38相比，基因转染对OVA-GMC38结肠癌细胞系致瘤能力没有影响。利用OVA免疫荷瘤小鼠，初步结果显示OVA-GMC38可用于评价结肠癌中针对OVA抗原的免疫治疗效果。

第二部分研究了负载模式抗原OVA的聚多巴胺纳米颗粒（OVA@PDA NPs）的表征和治疗功效。制备OVA@PDA NPs作为肿瘤疫苗，通过粒径分析仪及扫描电子显微镜观察纳米颗粒成均匀的、单分散的球形，细胞毒性实验表明纳米颗粒具有良好的生物相容性。共聚焦显微镜观察纳米颗粒吞噬发现纳米颗粒能够增加细胞对抗原的吞噬，并且被细胞内化的纳米颗粒可实现部分溶酶体逃逸。活体成像研究结果说明OVA@PDA NPs能够促进抗原在淋巴结处的聚集，并延长抗原滞留时间。而后的流式细胞术分析结果说明OVA@PDA NPs能够促进淋巴结内DCs表面成熟标志物的表达。建立OVA-GMC38结肠癌荷瘤小鼠模型，对荷瘤小鼠的免疫实验表明OVA@PDA NPs能够明显抑制肿瘤的生长，提高脾脏、淋巴结、肿瘤组织中CD4⁺、CD8⁺ T细胞的比例，促进了T细胞反应和T细胞增殖，改善肿瘤中免疫抑制的微环境。研究结果表明OVA@PDA NPs具有良好的免疫刺激作用，具有应用为肿瘤疫苗的潜力。

第三部分聚焦于负载肿瘤裂解物的聚多巴胺纳米颗粒（TCL@PDA NPs）的研究。通过多巴胺自聚合制备PDA NPs，再通过Michael加成和Schiff碱反应将肿瘤裂解物（TCL）共价连接到PDA NPs的表面上。TCL@PDA NPs的平均尺寸为241.9nm，多分散指数为0.200，具有良好的储存稳定性，对APC具有低的细胞毒性。此外，TCL@PDA NPs促进抗原摄取和DC成熟，导致DC细胞表面与抗原提呈相关的标志物的表达和细胞因子的分泌增强。用TCL@PDA NPs免疫荷瘤小鼠后，肿瘤进展显著延迟，细胞毒性CD8⁺ T细胞和记忆T细胞亚群显著增加，从而提供长期的抗肿瘤保护。此外，肿瘤内显著增多的CTL和明显锐减的M1型TAM以及骨髓来源的免疫抑制细胞提示TCL@PDA NPs具有突出的抗肿瘤能力。

总体而言，由于抗原加载方法简单、制备快速、有效的激活免疫和突出的抗肿瘤能力，作为抗原递送载体，PDA NPs是极具潜力的候选者。

with the development of surgical technique and chemotherapy, the treatment strategy of colorectal cancer (crc) continues to improve, but it still faces severe challenges in terms of treatment efficacy and side effects. recently, immunotherapy has been widely accepted, and has led to the breakthroughs of cancer treatment in some types of cancer, including colorectal cancer. at the same time, novel vaccines (nvs) are being developed with the potential to surpass traditional vaccines. polypeptide vaccine that is based on recombinant antigen or polypeptide is expected to become a substitute for traditional vaccine because of its good specificity and biosafety. on the other hand, polymer nanoparticles have outstanding advantages in tumor immunotherapy, because they possess capabilities of the high antigen loading, good biocompatibility and flexible modifiability. thus nanoparticle vaccine, a type of nvs, is attracting more and more attention. however, how to effectively activate anti-cancer immunity in vivo to achieve the purpose of tumor treatment still remains as a major challenge in the development of vaccines.

in this thesis, polydopamine nanoparticles (pda nps) with good biocompatibility and high loading capacity were used to load tumor model antigen ova or tumor lysate. we investigated their immunological evaluation in vitro and in vivo and tested their anti-tumor effect. the effect of pda nps loaded with single antigen and complex antigens on the anti-tumor immunity was evaluated with the colorectal cancer bearing mice, which provided basic experimental data and references for the application of pda nps in the tumor immunity field. the thesis is divided into the following three parts.

  the first part describes the construction of ova-gmc38 colon cancer cell line carrying tumor model antigen ova and green fluorescent protein (gfp). firstly, the lentivirus vector carrying double genes, ova and gfp genes, was designed and constructed. then puromycin was used to screen for positive ova-gmc38 cells among the lentivirus infected mc38 cells. the stable expression of gfp and ova genes in mc38 cells was detected by laser confocal microscope, flow cytometry, western blot and quantitative reverse tranase polymerase chain reaction (qrt-pcr). the effects on the establishment of tumor-bearing mice models with ova-gmc38 subcutaneous implantation in c57bl/6 mice were investigated. compared with mc38, the results showed that the transgene had no effect on the tumorigenic ability of ova-gmc38. using ova to immunize tumor-bearing mice, the preliminarily result showed that the ova-gmc38 can be applied to evaluate the immunotherapy efficacy against ova antigen in colorectal cancer.

  the second part investigated the characteristics and efficacy of pda nps loaded with model antigen ova (ova@pda nps). ova@pda nps was prepared as a tumor vaccine. observed under the particle size analyzer and scanning electron microscope, the nanoparticles appeared to be uniform and monodisperse spheres. cytotoxicity experiments showed that nanoparticles had good biocompatibility. by observation of the cellular phagocytosis of nanoparticles by confocal microscope, it was found that nanoparticles could enhance the phagocytosis of antigens, and a part of the nanoparticles that were internalized by the cells may escape from the lysosomes. in vivo imaging showed that ova@pda nps promoted the accumulation of antigens in lymph nodes and prolonged the retention time of antigens. the flow cytometry analysis showed that ova@pda nps could promote the expression of mature markers on the surface of dcs in lymph nodes. the tumor-bearing mouse model with ova-gmc38 colon cancer was established. the immunization experiment on tumor-bearing mice showed that ova@pda nps could significantly inhibit the tumor growth and increase the proportion of cd4⁺ and cd8⁺ t cells in spleen, lymph nodes and tumor tissues. ova@pda nps promoted t cell response and t cell proliferation. at the same time, ova@pda nps improved the microenvironment of intratumoral immunosuppression. the results showed that ova@pda nps has a good immunostimulatory effect and has the potential to be used as a tumor vaccine.

  the third part focused on the investigation of polydopamine nanoparticles (tcl@pda nps) loaded with tumor lysate. polydopamine nanoparticles were prepared by dopamine self-polymerization, and then the tumor lysate (tcl) was covalently connected to the surface of pda nps by michael addition and schiff base reaction. the average size of tcl @pda nps was 241.9 nm and the polydispersion index was 0.200. it had good storage stability and low cytotoxicity to bone marrow-derived dendritic cells (bmdc). in addition, tcl@pda nps promoted antigen uptake and dc maturation, resulting in increased expression of markers related to antigen presentation on the surface of dc cells and secretion of cytokines. after immunizing tumor-bearing mice with tcl@pda nps, the tumor progress was significantly delayed and the cytotoxic cd8⁺ t cells and memory t cell subsets were significantly increased, thus providing long-term protection against tumor. in addition to a significant increase in ctl, we observed a significant decrease in intratumoral m1 tam and bone marrow-derived immunosuppressive cells, which suggests that tcl @ pda nps has outstanding anti-tumor ability.

  in summary, as an antigen delivery carrier, pda nps is a promising candidate for antigen delivery because of its simple and rapid preparation, effective activation of immunity and outstanding anti-tumor ability.