多功能脂质体构建微针阵列疫苗佐剂-双传递系统

发布时间：2018-01-29 19:29

本文关键词： 微针阴道粘膜免疫应答佐剂系统脂质体　出处：《安徽医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：为发展有效的粘膜疫苗,本研究采用两种类型的多功能脂质体,一种为粒径为200nm的甘露糖化脂质A-脂质体(MLLs)和另一种粒径为50nm的隐形脂质A-脂质体(SLLs),两者均装载有模型抗原(OVA)及碳酸氢铵,并且组合在一起构建包含pro-SLLs/MLLs微针阵列(proSMMA)。研究表明,proSMMA再水化后迅速溶解,释放初始MLLs和SLLs。进一步的研究证明在阴道粘膜接种proSMMA后,再次释放的MLLs主要被阴道粘膜抗原提呈细胞所摄取,而SLLs则直接转运至引流淋巴结,被其中的抗原提呈细胞所摄取。此外,SLLs/MLLs能够促进抗原产生溶酶体逃逸,从而有望通过APCs(抗原提呈细胞)表面的MHC-I途径将抗原交叉提呈至淋巴细胞。产生溶酶体逃逸现象是由于溶酶体酸化脂质体使其释放NH4HCO3而生成CO2和NH4+/NH3再通过气体膨胀使得溶酶体破裂。体内追踪实验表明,SLLs将运载疫苗传递至淋巴结APCs,而MLLs将运载疫苗传递至接种粘膜APCs。因此,proSMMA实际上是疫苗佐剂-双递送系统,有望能够诱导体液和细胞免疫应答以预防通过性传播的病原体。本次实验以卵清白蛋白(Ovalbumin,OVA)为模型抗原,将OVA和碳酸氢铵共同装封在多功能脂质体中构建微针疫苗佐剂-双递送系统。结合微针给药机制,开展其作为经粘膜途径接种的疫苗递送系统。研究脂质体微针的制备及表征和多功能脂质体促进APCs对抗原的摄取,以及研究多功能脂质体对APCs功能影响及其部分机制等。通过分析脂质体微针经粘膜免疫应答特点,初步阐明其免疫应答效果。本研究主要分为两个部分,下面将对此两部分内容进行简述。第一部分:不同类型多功能脂质体微针阵列的构建及其表征。脂质体的制备方法分很多种,我们采用薄膜分散法成功制备了两种多功能脂质体SLLs和MLLs,并且同微针基质相关物质混合通过微针模具成功制备了微针阵列。在后续实验中为追踪脂质体在体内及细胞内的趋向,还制备了装载荧光染料钙黄绿素和磺酰罗丹明B(SRB)的脂质体。随后通过对脂质体粒经,zeta电位,结合效率等方面进行检测,从而完成对脂质体及proSMMA的表征。第二部分:不同类型多功能脂质体微针阵列的部分免疫机制考察,其中探讨了微针经粘膜接种的主要机制,包括通过共聚焦显微镜观察脂质体被APCs摄取后的细胞内定位,通过流式细胞术考察脂质体刺激APCs活化成熟以及抗原提呈,再将所制备的proSMMA通过阴道粘膜途径接种后分析俩种脂质体的体内趋向。
[Abstract]:For the development of effective mucosal vaccine, this research adopts the multifunctional liposome two types, a particle size of mannosylated lipid A- 200nm liposomes (MLLs) and stealth liposome lipid A- a diameter of 50nm (SLLs), both of which are loaded with antigen (OVA) and model ammonium hydrogen carbonate, and together construct containing the pro-SLLs/MLLs micro needle array (proSMMA). The results show that proSMMA after rehydration of rapid dissolution, release of initial MLLs and SLLs. further demonstrated in the vaginal mucosa after inoculation with proSMMA, the MLLs is mainly released again vaginal mucous membrane antigen presenting cell uptake, and SLLs transfer directly to the draining lymph nodes, which are antigen-presenting cell uptake. Furthermore, SLLs/MLLs can promote the antigen of lysosomal escape, which is expected to pass APCs (antigen presenting cell surface antigen) MHC-I pathway cross presentation to lymphocytes. Have lysosomal escape phenomenon is due to the release of lysosomal acidification liposome NH4HCO3 and formation of CO2 and NH4+/NH3 by gas expansion. The lysosomal rupture in vivo tracking experiments show that SLLs will launch vaccine delivery to the lymph nodes of APCs, and MLLs will launch vaccine delivery to the mucosal vaccination APCs. so that proSMMA is actually a vaccine adjuvant - delivery systems. Is expected to be able to induce humoral and cellular immune responses to prevent sexually transmitted pathogens. The experiment with egg albumin (Ovalbumin, OVA) as a model antigen, OVA and ammonium bicarbonate common sealed micro needle vaccine adjuvant - build delivery system in multifunctional liposome. Combined with micro needle mechanism. To carry out the mucosal vaccination ways as vaccine delivery system. And the characterization of the micro needle of liposome preparation and multifunctional liposome to promote the uptake of antigen APCs, and Research on multi function Liposome on APCs function and its influence mechanism. Through the analysis of the liposome micro needle through the mucosal immune response characteristics, clarify its immune response. This research is mainly divided into two parts, the following will be two parts briefly. The first part: different types of multifunctional liposome construct micro needle array and characterization. Liposome preparation methods are many points, we use two kinds of thin film multifunctional liposome SLLs and MLLs dispersion were successfully prepared, and related with the micro needle matrix material mixed by micro needle mould were successfully prepared. The micro needle array to track the trend of liposomes in vivo and in subsequent cells in the experiment, also prepared a loaded fluorescent dye calcein and sulfonyl Luo Danming B (SRB) liposome. Then based on the liposome particle, zeta potential, combined with the efficiency and other aspects of detection, thus completing the liposome and proSMMA The second part: the research of characterization. The immune mechanism of different types of multifunctional liposome micro needle array, which explores the main mechanism of the micro needle by mucosal vaccination, including through confocal microscopy by APCs liposome after ingestion of the cellular localization, through liposome stimulated APCs activation and maturation of antigen-presenting flow cells were then analysis on the trend of two kinds of liposomes in vivo prepared proSMMA inoculated by the vaginal mucosa. After the way

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R945

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