聚乳酸微球对乙肝表面抗原的吸附及其细胞免疫应答规律
文献类型：学位论文
| 作者 | 李娜 |
| 学位类别 | 硕士 |
| 答辩日期 | 2010-06-04 |
| 授予单位 | 中国科学院研究生院 |
| 导师 | 苏志国 ; 马光辉 |
| 关键词 | 快速膜乳化 乙肝表面抗原 佐剂 聚乳酸微球 细胞免疫应答 |
| 其他题名 | Adsorption of hepathtis B surface antigen on PLA microspheres and elucidation of cellular immune response by the novel therapeutic |
| 学位专业 | 生物化工 |
| 中文摘要 | 在乙型肝炎的治疗过程中，宿主免疫系统针对病毒所产生的免疫应答非常重要。已有的乙肝疫苗仅能起到预防作用，但没有治疗作用。以聚乳酸（Polylactic, PLA）微球作为佐剂有望增强抗原提呈细胞的吞噬能力，进而有望增强抗原诱导的细胞免疫应答。本论文制备了粒径均一的PLA微球，以其作为佐剂吸附乙肝表面抗原（HBsAg），快速诱导细胞免疫应答，以期开发出治疗性乙肝疫苗制剂。 首先采用不同的膜乳化技术和溶剂去除法制备尺寸均一的PLA微球，以其作为乙肝表面抗原的佐剂。然后采用不同表达系统的HBsAg、不同粒径和材料的微球佐剂以及不同微球负载抗原方式，对特异性诱导机体细胞免疫反应方面的差别进行了比较，发现小于1 μm的小粒径PLA微球吸附汉逊HBsAg能够显著增强HBsAg诱导的IFN-γ分泌水平。考察了微球膜材和粒径对HBsAg体外吸附行为、活性和结构的影响，发现疏水性PLA微球对HBsAg的吸附率最大，PLGA微球对HBsAg活性保留最高；微球粒径越小，HBsAg吸附率越大，而HBsAg的活性保留没有明显变化。以高HBsAg吸附率、高HBsAg活性和最佳细胞免疫效果为目标，系统考察了不同吸附条件对HBsAg体外和体内活性的影响，获得了最优吸附条件。优选条件制备的微球乙肝疫苗能同时显著增强HBsAg诱导的IFN-γ分泌水平和特异性CTL杀伤作用，呈现出较强的佐剂作用，而以铝盐为佐剂的商品化疫苗尽管对HBsAg诱导的IFN-γ分泌有一定的增强作用，但却抑制了HBsAg诱导的特异性CTL杀伤作用；在体液免疫方面，以PLA微球为佐剂的抗原制剂能达到铝佐剂疫苗相似的效果。利用亲水阳离子聚合物对PLA空白微球进行表面修饰，修饰后PLA微球表面的正电荷逐渐增加，显著提高了PLA微球对HBsAg的吸附，并能够在一定程度上增强微球的佐剂效应。 研究表明，用聚乳酸微球作为乙肝疫苗佐剂，可以体现出较好的增强抗原诱导的特异性细胞免疫反应的佐剂作用，为乙肝治疗性疫苗的研究提供了一个新的思路。 |
| 英文摘要 | The antiviral immune response of the host plays a vital role in the course of hepatitis B virus (HBV) infection therapy. The hepatitis B vaccines currently available in market are the effective prophylactic vaccines, which do not work therapeutically. The microspheres as adjuvants would be ideal for passive targeting of antigen presenting cells, and active specific T cells to induce high antigen specific cellular immunization response. The objectives of this thesis are to prepare uniform-sized polylactide (PLA) microspheres, as well as to investigate the application of PLA microspheres as adjuvants for novel hepatitis B therapeutic vaccine. The uniform-sized PLA microspheres with different particle size were prepared by membrane emulsification technique, which would be used as adjuvants of HBsAg. The PLA microspheres’ adjuvanticity in enhancing the cellular immune response induced by HBsAg was primarily studied. It was found that HBsAg expressed by Hansenula polymorpha and PLA microspheres with a diameter under 1 μm had shown potentials in inducing specific IFN-γ secretion than others. An effective therapeutic vaccine was developed by adsorbing HBsAg on the surface of PLA microspheres. The effects of polymer composition and microspheres diameter on the adsorption efficiency, bioactivity retention and structure of HBsAg were investigated. The hydrophobic PLA microspheres possessed higher adsorption efficiency, and the PLGA microspheres owned higher bioactivity retention of HBsAg. The adsorption efficiency of HBsAg decreased with the increase of diameter of microspheres, whereas which had little influence on the bioactivity of HBsAg. The adsorption conditions were optimized for obtaining better cellular immunity with higher adsorption rate and bioactivity of HBsAg, and the immune response of as-prepared therapeutic hepatitis B vaccine was studied. It was found that the microspheres vaccine could effectively stimulate the mice splenocytes to secret HBsAg-specific IFN-γ, and the levels of IFN-γ secretion were far higher than commercial alum vaccine. The results of cytolytic assays also demonstrated that microspheres vaccine could enhance the cytotoxic activity induced by HBsAg. In addition, the level of anti-HBsAg titer obtained from as-prepared hepatitis B vaccine was the same or higher than that of alum-vaccine. The surface of PLA microspheres was modified by adsorption of oppositely charged polyelectrolytes, and the effect of the surface charge of microspheres on the activation of cellular immune response was investigated. It was found that the presence of polycations shells around the microspheres facilitated the adsorption of HBsAg, and enhanced the adjuvanticity of PLA microspheres. In conclusion, the uniform-sized PLA microspheres have great potentials as adjuvant for hepatitis B therapeutic vaccine, which provided a new strategy for hepatitis B therapeutic vaccine. |
| 语种 | 中文 |
| 公开日期 | 2013-09-17 |
| 页码 | 132 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1505]  |
| 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 | 李娜. 聚乳酸微球对乙肝表面抗原的吸附及其细胞免疫应答规律[D]. 中国科学院研究生院. 2010. |
入库方式： OAI收割
来源：过程工程研究所
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