基于肿瘤抗原的纳米疫苗制备、评价及抗肿瘤效果研究

发布时间：2018-01-15 11:38

本文关键词：基于肿瘤抗原的纳米疫苗制备、评价及抗肿瘤效果研究　出处：《北京协和医学院》2017年博士论文　论文类型：学位论文

【摘要】：研究目的:尽管全肿瘤细胞裂解物已经被作为肿瘤抗原的来源用于癌症疫苗的研发,但是目前基于肿瘤细胞裂解物制备树突状细胞疫苗的抗肿瘤免疫治疗的临床试验结果不容乐观。肿瘤细胞裂解物作为肿瘤抗原制备肿瘤免疫治疗疫苗的疗效尚有较大的改进空间。因此,通过这种方式制备的肿瘤疫苗诱导的抗肿瘤免疫反应的新技术和方法有待进一步增强。为了改善基于肿瘤细胞裂解物疫苗的治疗效果,生物材料在提高抗原递送和提呈等方面具有很重要的作用。本研究构建了一种以壳聚糖纳米粒为基础,具有甘露糖受体靶向的新型还原响应多糖纳米载体,通过静电吸附作用实现与肿瘤细胞裂解物结合形成纳米粒疫苗,能够有效提呈肿瘤抗原和活化T细胞,并通过引入靶向树突状细胞(Dendritic cells,DCs)的官能基团和佐剂增强肿瘤抗原的免疫原性以及机体对疫苗的免疫应答效果。方法:首先将壳聚糖与肿瘤细胞裂解物通过静电吸附方式结合,同时合成甘露糖修饰的海藻酸钠(Man-ALG),通过静电吸附方式与壳聚糖负载的肿瘤细胞裂解物结合形成纳米粒疫苗制备甘露糖修饰的壳聚糖负载肿瘤细胞裂解物纳米粒(Man-CTS-TCLNPs)。通过核磁共振氢谱(1H-NMR)和红外光谱(IR)来表征化学结构,透射电镜(TEM)确定结构;利用BCA法测定在不同的pH值下Man-CTS-TCLNPs中蛋白的释放情况;CCK-8法以及LDH法评价纳米粒对骨髓来源的树突状细胞(BMDC)的毒性;通过激光共聚焦以及流式细胞仪检测BMDC对Man-CTS-TCL NPs的摄取并进行定性和定量分析;体内外评价Man-CTS-TCL NPs对骨髓来源的树突状细胞促成熟能力,流式细胞仪检测与BMDC成熟相关的表面标志的表达情况,ELISA法检测炎症相关细胞因子的分泌情况;利用小动物活体成像系统考察皮下注射Man-CTS-TCL NPs后其在体内的分布情况以及向回流淋巴结的迁移能力;通过三种不同的动物模型(预防性模型、治疗性模型以及肺转移模型)来研究这种新型的纳米粒疫苗的抗肿瘤效应,并探讨其作用机制。结果:我们成功地制备了 Man-CTS-TCLNPs纳米疫苗,并通过1H-NMR和IR确定了其结构。TEM结果显示Man-CTS-TCLNPs具有特征性的球型结构,在pH为5.0时能够更持久的释放抗原。壳聚糖负载肿瘤细胞裂解物纳米粒(CTS-TCLNPs)和Man-CTS-TCL NPs可以有效地被BMDC细胞摄取,毒性实验结果显示Man-CTS-TCL NPs具有良好的生物学相容性。Man-CTS-TCL NPs组的细胞摄取明显增加,体内更具有抗原提呈细胞靶向性,皮下注射后24 h主要分布在回流淋巴结。Man-CTS-TCL NPs可以增强树突状细胞表面标志CD80、CD86、CCR7、MHCI、MHCII以及CD40的表达,相应的细胞因子IFN-γ、IL-4等的表达也上调。预防性动物模型结果显示,免疫的纳米粒疫苗组小鼠能够延缓肿瘤的出现,具有较好的抑制肿瘤生长效果。治疗性动物模型结果显示,与其他组相比,Man-CTS-TCL NPs治疗组,肿瘤的体积以及重量明显的减小,具有统计学差异。荷瘤小鼠用纳米粒疫苗免疫后,具有增强的CTL反应,且小鼠体内的抗TCL特异性抗IgG抗体水平也显著提高,小鼠血清中IFN-γ的分泌明显增强。荷瘤鼠肺部转移模型结果显示,Man-CTS-TCL NPs通过尾静脉注射后,能够抑制黑色素瘤细胞向肺部的转移情况,这种效果可能是通过活化小鼠外周血中的细胞毒性T细胞发挥杀伤作用来完成的。结论:总之,我们成功的制备了壳聚糖纳米粒,构建了甘露糖修饰的壳聚糖纳米粒负载肿瘤细胞裂解物疫苗,这种疫苗具有较低的细胞毒性,更容易被抗原提呈细胞摄取,能够靶向到体内的抗原提呈细胞并可在回流淋巴结中聚集,体内外结果显示能够诱导未成熟DC细胞的成熟,促进细胞因子的释放,体内的预防性以及治疗性实验证明了此纳米粒疫苗具有良好的预防黑色素瘤形成以及抑制肿瘤生长和转移的效果。在肺转移模型中,能够抑制黑色素瘤细胞向肺部的转移情况。本课题成功的设计了新型的靶向树突状细胞的壳聚糖纳米粒疫苗,为探索基于纳米生物材料的抗肿瘤免疫疗法提供了一定的理论基础。
[Abstract]:Objective: Although the whole tumor cell lysates were used as source of tumor antigens for cancer vaccine development, but at present the tumor cell lysate was prepared based on the anti tumor immunity of dendritic cell vaccine for clinical trials is not optimistic. Tumor cell lysate as a tumor antigen preparation of tumor immunotherapy vaccine efficacy is larger improvement of space. Therefore, through the new technology and method of the anti-tumor immune response induced by tumor vaccine prepared by the need to be further enhanced. In order to improve the tumor cell lysate vaccine treatment based on biological material plays a very important role in improving antigen delivery and presentation. This research constructs a chitosan nanoparticles as the foundation, has the mannose receptor targeted model reduction response polysaccharide nanoparticles and by electrostatic adsorption Combined with tumor cell lysate to form nanoparticles vaccine can effectively present tumor antigen and activation of T cells, and dendritic cells by introducing the target (Dendritic cells, DCs) immune tumor antigen immunogenicity and vaccine response to the body effect of the functional groups and adjuvants. Methods: firstly, chitosan and tumor cells lysates by electrostatic adsorption method combined with the simultaneous synthesis of mannose modified sodium alginate (Man-ALG), combined with tumor cell lysate by electrostatic adsorption and the formation of chitosan supported nanoparticles vaccine preparation of mannose modified chitosan nanoparticles loaded with tumor cell lysate (Man-CTS-TCLNPs). By 1H NMR and IR (1H-NMR) the spectrum (IR) to characterize the chemical structure, transmission electron microscopy (TEM) to determine the release of protein structure; Determination of Man-CTS-TCLNPs in different pH by BCA method; CCK-8 Dendritic cell evaluation method and LDH method of nanoparticles derived from bone marrow (BMDC) toxicity; through the uptake of Man-CTS-TCL NPs laser confocal and flow cytometry BMDC for qualitative and quantitative analysis; in vitro and in vivo evaluation of Man-CTS-TCL NPs on bone marrow derived dendritic cell maturation promoting ability, expression of surface marker detection and BMDC flow cytometry mature related to the secretion of inflammatory cytokines detected by ELISA; using small animal in vivo imaging system of subcutaneous injection of Man-CTS-TCL NPs after the in vivo distribution and migration to draining lymph nodes; by three different animal models (model and lung metastasis model of preventive model. Anti tumor effect of treatment) to study this novel nanoparticle vaccine, and explore its mechanism. Results: we successfully prepared Man-CTS-TCLNPs nano preparation M vaccine, and through 1H-NMR and IR to determine the structure of.TEM results show that the spherical structure has characteristic Man-CTS-TCLNPs, can release more durable antigens at pH 5. Tumor cell lysate loaded chitosan nanoparticles (CTS-TCLNPs) and Man-CTS-TCL NPs can effectively be BMDC cell uptake, toxicity test results showed that cellular uptake Man-CTS-TCL NPs has good biological compatibility of.Man-CTS-TCL in NPs group increased significantly, the body has more antigen-presenting cell targeting, after subcutaneous injection of 24 h mainly distributed in the draining lymph nodes of NPs.Man-CTS-TCL to enhance the dendritic cell surface markers CD80, CD86, CCR7, MHCI, MHCII and CD40 expression of cytokines IFN- accordingly, also raised the expression of IL-4. Prophylactic animal model showed that mice immune vaccine nanoparticles can delay the appearance of tumor, inhibit the swelling is good The effect of treatment of tumor growth. The animal model showed that, compared with the other groups, Man-CTS-TCL NPs group, the tumor volume and weight were significantly decreased, with statistical difference. The tumor bearing mice after vaccination with nanoparticles, with enhanced CTL reaction, and mouse anti TCL specific IgG antibody levels increased significantly and secretion of mouse serum levels of IFN- increased obviously. The tumor bearing mice lung metastasis model results show that Man-CTS-TCL NPs through tail vein injection, can inhibit melanoma cell metastasis to the lungs, this effect may be through the activation of cytotoxic T cells in peripheral blood of mice in the play killing effect to complete. Conclusion: in summary, we successfully prepared the chitosan nanoparticles, constructed the mannose modified chitosan nanoparticles loaded with tumor cell lysate vaccine, this vaccine has low cytotoxicity, More likely to be antigen-presenting cell uptake, can be targeted to the body of antigen-presenting cells and can accumulate in draining lymph nodes, in vitro and in vivo results showed that it can induce immature DC cells to mature, to promote the release of cytokines in vivo, preventive and therapeutic experiment show that this vaccine can prevent melanoma nanoparticles the formation and inhibition of tumor growth and metastasis. In lung metastasis model, can inhibit melanoma cell metastasis to the lungs. This study successfully designed a novel targeted dendritic cell chitosan nanoparticles vaccine for exploration provides a theoretical basis of anti tumor immunotherapy nanobiomaterials based on.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R943;R96

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