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题名	纳微米粒子作为疫苗佐剂的研究
作者	岳华
学位类别	博士
答辩日期	2012-05-22
授予单位	中国科学院研究生院
导师	苏志国
关键词	佐剂 粒径效应 免疫应答 纳微米粒子 氧化石墨烯
其他题名	Exploration of the Nano- to Micro-particles as Vaccine Adjuvants
学位专业	生物化工
中文摘要	纳微米粒子在药物传输、肿瘤诊断、分子成像以及疫苗递送方面展现出巨大的应用前景。虽然纳微米粒子性质（如大小、形状和电荷性质）对生物学效应的影响已引起广泛关注，但是目前对于以纳微米粒子作为疫苗佐剂时还缺乏研究及相应的理论指导。本论文对这一全新的领域进行了探索，制备具有不同粒径和不同维度的纳微米粒子，研究其作为疫苗佐剂时对免疫效果的影响。首先，以壳聚糖纳微米球作为三维球型颗粒模型，对纳米球佐剂的可行性进行了论证，并在此基础上对聚乳酸纳米球作为治疗性乙肝疫苗佐剂的应用进行了探索。此外，从不同维度层面上，考察了二维平面粒子氧化石墨烯的粒径效应，并进一步探讨了其作为疫苗佐剂时对免疫学效果的影响。 论文具体开展的研究工作如下： 1．以壳聚糖纳微米球颗粒为佐剂模型，评价了粒径大小对细胞学、免疫学效应的影响。通过使用膜乳化技术和特定的交联方法，制备出粒径均一的、具有自发荧光特性的颗粒，研究了粒径对巨噬细胞吞噬能力、内化机制、胞内运输和细胞因子水平等方面的影响。结果表明，与微米球相比，纳米球更容易被细胞大量而快速地摄取，并且具有更大的内吞颗粒表面积，更适合作为有效的抗原吸附和运输载体；并且，纳米球可以通过溶酶体途径被运输，理论上可以使携带的外源性抗原更充分地被加工；此外，相比微米球，纳米球有助于提高免疫激活相关的细胞因子分泌水平，而下调免疫抑制答相关的细胞因子分泌水平。因此，具有较小粒径的纳米颗粒在作为疫苗佐剂时具有潜在的优势。 2．在上述壳聚糖纳微米作为颗粒佐剂模型并确定了最佳粒径的基础上，探索了聚乳酸（PLA）纳米球在治疗性乙肝疫苗中应用的可行性。通过将粒径均一的PLA纳米球与乙肝表面抗原复配后制得疫苗制剂，从细胞和动物水平上考察了其作为疫苗佐剂的潜力。实验结果表明聚乳酸（PLA）纳米颗粒体系能够促进乙肝表面抗原的摄取并以多种方式提呈，提高了抗原提呈细胞（APCs）共刺激因子的表达，并上调了CD4+ T和CD8+ T淋巴细胞的识别信号分子。在抗原摄取和提呈能力同时得到提升的基础上，纳米球佐剂不仅超越了铝佐剂在诱导抗体滴度方面的优势，并且有效上调了评价细胞免疫应答的两个关键指标（干扰素分泌水平和特异性裂解作用），最终实现体液免疫和细胞免疫双重应答的效果。PLA纳米球作为治疗性乙肝疫苗佐剂的巨大优势，有望打破乙肝病毒导致的免疫耐受。 3．在上述三维球形颗粒的基础上，考察了二维新型碳材料氧化石墨烯（GO）不同粒径对细胞学、免疫学效应的影响。通过对GO内在特质的探索，解决了粒径不均一和胞内难以检测的问题，成功地在多种细胞系中对GO的粒径影响进行了全面系统评价。与传统球形颗粒不同，平面GO粒子的内吞存在严格的细胞种属选择性，只有具有吞噬功能的细胞才可以将其摄取。此外，在片层数相同的情况下，GO的内化没有粒径依赖性，巨噬细胞对350 nm和2 μm GO粒子的摄取量一致，这种特殊的现象主要与Fcγ受体介导的吞噬作用有关。虽然粒径对细胞的摄取能力没有影响，但是显著影响GO与细胞之间的相互作用、在胞内的定位情况以及巨噬细胞炎性因子的分泌情况。与350 nm GO相比，2 μm GO可以分泌与免疫激活有关的细胞因子或者趋化蛋白（如IL-12、IFN、TNF、MCP和IL-6），并促使炎症的发生，在作为疫苗佐剂时可能具有更独到的优势。 4.利用模式抗原卵清蛋白（OVA）和转基因小鼠模型OT-1，系统评价了2 μm GO在疫苗佐剂中的应用。GO对OVA抗原的摄取在初始阶段有明显的促进作用，并且其表面负载的抗原在胞内的释放和作用时间相比单纯抗原显著延缓，显示出“抗原储蓄库”的效应。GO为T细胞的激活提供了持续而稳定的T细胞识别信号（MHC I和MHC II）以及激活信号（CD80/86），并且延长了OVA特异性“MHC I-抗原肽”在APCs表面展示的时间。另外，GO-OVA还可以促进APCs和T细胞分泌与免疫激活有关的细胞因子。在双信号分子和细胞因子的共同作用下，GO-OVA抗原制剂有效地促使抗原交叉提呈给OVA特异性CD8+ T细胞，并促进其发生特异性增殖，为以诱发细胞免疫应答为目的新型疫苗佐剂的开发提供了理论依据。
英文摘要	To date, micro/nano particles (MP/NP) have shown promising signs in biomedical applications, including drug delivery, tumor diagnosis, molecule imaging, and vaccine adjuvant. A deeper understanding of how the physiochemical properties (e.g. size, shape, and charge) of particles regulate specific biological responses is becoming a crucial requirement for their successful application. However, this research area is still in its infancy, especially when using micro/nano particles as vaccine adjuvant. To provide insights on the design of particulate adjuvant, we prepared particles with different size and dimensions and evaluated their biological response on aspect of vaccine application. As for the spherical particles, the feasibility of exploiting these particles as the vaccine adjuvant was clarified, by using the chitosan particles as the phenotype. On the basis of these theoretical results, we successfully employed the polylactide (PLA) NP as the adjuvant in the therapeutic vaccine. As for the flat particles, the size effect of the unique two-dimensional (2D) material (graphene oxide, GO) on the cellular response was investigated from the view of particle dimensions. In terms of these results, we further assayed the adjuvanticity of microsized GO by using a model antigen. In detail, this thesis mainly included the following issues: 1. We evaluated the size effects of spherical particles on a series of cellular responses. The uniform-sized and autofluorescent chitosan MP/NP were prepared by using the porous membrane emulsification technique and specific crosslink reaction to investigate the effect of particle size on macrophage responses, including cellular uptake, cellular internalization mechanism, intracellular trafficking, and cytokine profile. In comparison with the MP, the NP accumulated in the cells at a faster rate and with a higher surface area that was preferable for drug adsorption and conjunction. Furthermore, the NP transported via lysosomal pathway, which was capable of being degraded and presented extensively. In addition, the NP preferred to promote the cytokine secretion of immune stimulators rather than immune suppressors. Therefore, the particles in nanosize were superior to be employed as adjuvants in vaccination. 2. On the basis of aforementioned investigations, we successfully explored the PLA NPs as the therapeutic vaccine adjuvant for hepatitis B treatment. The NP-based antigen formulation that obtained by mixing the hepatitis B surface antigen (HBsAg) and the uniform sized PLA NP was used to assess their immunological performances on provoking HBV-specific immune response. In vitro studies showed that HBsAg accumulated in antigen-presenting cells (APCs) to a larger content with the assistant of NP than that of the pure antigen-group. Besides the elevated recognition signal (major histocompatibility complex, MHC) for both CD4+ T and CD8+ T, the costimulators (CD80/86) were also found upregulated. These results were mostly owing to the divergent antigen trafficking ways of NP-antigen in APCs. Further in vivo experiments established that NP-antigen not only surpassed the commercialized Alum-antigen in the upregulation of specific antibody titer, but also enhanced the cellular response indicated by the higher CD8+ CTL cytotoxicity and IFN-γ cytokine secretion. These results strongly supported that NP-based antigen could promote an orchestration of cellular and humoral response, exhibiting favorable intrinsic properties to be applied in therapeutic vaccines. 3. Having revealed the size effect on traditional spherical particles, we continued to investigate the size effect of the 2D flat GO on the cellular response and immunological issues. We separated the GO sheets in different size and systematically investigated the GO in response to different types of cells. In terms of abilities to internalize GO, enormous discrepancies were observed in the six cell types, with only the phagocytes found to be capable of internalizing GO. The 2 μm and 350 nm GO greatly differed in lateral dimensions, but equally contributed to the uptake amount in macrophages. Similar amounts of antibody opsonization and active Fcγ receptor-mediated phagocytosis were demonstrated the cause of this behavior. Although the GO size contributed little on the cellular internalization, the 2 μm GO showed divergent intracellular locations and induced much stronger inflammation related responses. The microsized GO promoted the secretion of cytokines (IL-12, IFN, TNF, MCP, and IL-6) that related to the immune activation. Since macrophages belong to the APCs, these assays might provide insights into the potent of 2 μm GO as vaccine adjuvant. 4. By employing the model antigen (ovalbumin, OVA) and the corresponding transgene mice model (OT1), we further assessed the adjuvant capacity of 2 μm GO. In the initial uptake stage, the OVA internalization was significantly facilitated with the assistant of GO, and the loaded antigen was released in a slow and continuous manner, acting as an antigen reservoir. In comparison with pure antigen, GO-OVA provided stable and sustained recognition signals (MHC I and MHC II) and costimulator signals (CD80/CD86), and prolonged the exhibition time of the OVA specific “MHC-antigen peptide” on the surface of APCs (dendritic cells and macrophages). Moreover, the GO-OVA formulation could benefit the production of immune stimulator cytokines in both APCs and T cells. Taking advantage of these two signals and the cytokines, the proliferation of OVA specific CD8+ T cells, which required for the cellular immunity, was efficiently promoted by GO. This work thus helped build useful knowledge for incorporation of the unique 2D nanomaterial as vaccine adjuvant.
语种	中文
公开日期	2013-09-25
内容类型	学位论文
源URL	[http://ir.ipe.ac.cn/handle/122111/1844]
专题	过程工程研究所_研究所（批量导入）
推荐引用方式 GB/T 7714	岳华. 纳微米粒子作为疫苗佐剂的研究[D]. 中国科学院研究生院. 2012.

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