弓形虫exosomes的生物学特性及其调节宿主免疫功能的研究

发布时间：2018-01-18 06:12

本文关键词：弓形虫exosomes的生物学特性及其调节宿主免疫功能的研究　出处：《山东大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景:弓形虫是一种专性细胞内寄生的原虫,呈世界性分布,可感染包括人和牲畜在内的所有温血动物,引起人兽共患病的弓形虫病,严重威胁人类健康和畜牧业发展。目前,尚无理想的药物用来防治弓形虫病。因此,深入研究弓形虫的致病机制,寻找新的防治弓形虫病的药物或疫苗显得尤为重要。Exosomes是有核细胞分泌到细胞外环境的膜性小囊泡,直径在30-100 nm之间。其中病原体分泌的exosomes具有一些特殊的生物学功能,如介导病原体与宿主的信息交流、调节宿主免疫应答等。然而,弓形虫是否产生并分泌exosomes尚不清晰。研究目的:从弓形虫RH株和ME 49株的速殖子培养上清液中分别提取并鉴定exosomes,评价弓形虫exosomes对巨噬细胞和BALB/c小鼠的免疫调节作用,为寻找防治弓形虫病的药物或疫苗提供实验依据。研究方法:利用透射电镜和Western blotting分析鉴定从弓形虫速殖子培养液中获取的exosomes形态结构和表面标志。用PKH 67绿色荧光染料对弓形虫分泌的exosomes进行染色后与RAW264.7巨噬细胞共孵育,在荧光显微镜下观察exosomes与巨噬细胞融合情况,采用ELISA法检测弓形虫exosomes影响小鼠巨噬细胞分泌细胞因子水平。将弓形虫exosomes经肌肉注射免疫接种BALB/c小鼠,检测小鼠体内产生的体液免疫与细胞免疫应答来评价其作为抗弓形虫候选疫苗的免疫保护效果。结果:透射电镜和WB检测结果显示,弓形虫exosomes形态结构呈圆形或椭圆形、直径大小在10-150 nm之间、含有蛋白分子P30和Hsp 70。体外实验结果显示,弓形虫exosomes能与巨噬细胞融合,且能调节巨噬细胞分泌细胞因子的水平。弓形虫exosomes免疫接种BALB/c小鼠后能产生更高水平的IFN-γ、IL-12、IgG和IgG2a,小鼠的生存时间也明显延长。体内实验结果显示,弓形虫exosomes诱导小鼠产生一定的体液免疫与细胞免疫,可为抵抗弓形虫速殖子攻击提供部分免疫保护力。结论:弓形虫exosomes与巨噬细胞融合并调节其分泌细胞因子水平,表明巨噬细胞是弓形虫exosomes的靶细胞之一。弓形虫分泌的exosomes作为疫苗可刺激小鼠产生一定的体液免疫和细胞免疫应答,表明弓形虫exosomes也可成为抵抗细胞内病原体的候选疫苗,为研制弓形虫新型疫苗奠定理论基础。
[Abstract]:Background: Toxoplasma gondii is an obligate intracellular parasitic protozoa, with worldwide distribution, can infect all warm blooded animal including human and livestock, causing zoonoses toxoplasmosis, a serious threat to human health and animal husbandry development. At present, there is no ideal drug for prevention of toxoplasmosis. Therefore, pathogenic mechanism study of Toxoplasma gondii, looking for new drugs or vaccines for prevention of toxoplasmosis is particularly important to.Exosomes is a membranous capsule extracellular environment secretted mononuclear cells, 30-100 in diameter between nm. The pathogens secrete exosomes has some special biological functions, such as information exchange mediated by pathogen and host, regulation the host immune response. However, whether the production and secretion of exosomes of Toxoplasma gondii is unclear. Objective: are supernatant from Toxoplasma gondii RH strain and ME strain 49 tachyzoites culture The evaluation and identification of exosomes, regulation of immunity of Toxoplasma gondii exosomes on macrophage and BALB/c in mice, and provide experimental basis for prevention drugs for toxoplasmosis or vaccine. Methods: using transmission electron microscope and Western analysis and identification of blotting from Toxoplasma gondii tachyzoites in the culture medium to obtain exosomes morphology and surface markers on the secretion of Toxoplasma gondii. PKH 67 green fluorescent dye exosomes were stained with RAW264.7 macrophages incubated with exosomes macrophage fusion observed under fluorescent microscope. ELISA method was used to detect Toxoplasma gondii exosomes mice macrophage cytokine secretion. The exosomes of Toxoplasma gondii by intramuscular immunization of BALB/c mice in mice generated humoral immunity and to evaluate the cellular immune response as immune protective effect against Toxoplasma gondii vaccine candidates. Results: through Transmission electron microscopy and WB assay showed that the morphology of exosomes from Toxoplasma gondii were round or oval in diameter 10-150 between nm, P30 and Hsp 70. protein molecules in vitro experiments showed that Toxoplasma exosomes and macrophage fusion, and regulation of macrophage cytokine secretion level. IFN- gamma, exosomes of Toxoplasma gondii BALB/c immunization after the mice had higher levels of IL-12, IgG and IgG2a, the survival time of mice was significantly prolonged. In vivo results showed that humoral immune and immune cells of Toxoplasma gondii in mice induced by exosomes, can provide some protective immunity against Toxoplasma attack. Conclusion: Toxoplasma gondii exosomes and macrophages fusion and adjusting the level of secretory cytokines, suggesting that macrophages are target cells of Toxoplasma gondii exosomes. Toxoplasma gondii exosomes as vaccine can stimulate the secretion of mice Certain humoral and cellular immune responses are generated, indicating that Toxoplasma gondii exosomes can also be a candidate vaccine against intracellular pathogens, which lays a theoretical foundation for developing new vaccine of Toxoplasma gondii.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R382.5

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