禽流感灭活病毒诱导鼻腔免疫应答机制的研究

发布时间：2018-04-21 16:37

本文选题：禽流感灭活全病毒 + 鼻腔黏膜　；参考：《南京农业大学》2015年博士论文

【摘要】：禽流感(Aivan Influenza,AI)已给我国和世界上许多国家的养禽业造成了极大的经济损失。禽流感主要分为高致病性和低致病性禽流感。虽然低致病禽流感的发病和死亡率均较低,但宿主可以作为携带者传播病毒,为流感病毒变异重组提供了资源库。鼻腔是禽流感病毒进入机体的主要入口之一。研究表明经鼻腔免疫可以在局部建立有效的黏膜免疫应答,直接切断病毒的感染路径。然而,由于鼻腔黏膜屏障的阻碍,单独应用灭活流感病毒进行鼻腔免疫时不能有效地提升机体免疫力。近年来,CpG ODN(CpG Oligodeoxynucleotides)佐剂因其具有较强的免疫刺激作用,已受到广泛关注和研究。CpG ODN的主要作用机制是可以靶向并促进黏膜下树突状细胞(Dendritic cells,DCs)成熟,进而有效地增强黏膜和系统免疫应答。但是,在黏膜屏障存在的情况下,CpG ODN是否有助于禽流感灭活病毒颗粒的跨鼻腔黏膜递送至今不明确。因此,本研究首先确证CpG ODN配合H9N2禽流感灭活全病毒(H9N2 WIV)鼻腔免疫小鼠后局部黏膜免疫和系统免疫应答水平。再次,在体外试验中,CpG ODN配合H9N2 WIV直接与小鼠DCs(体外诱导培养)相互作用,应用流式细胞术、ELISA试验和混合淋巴细胞反应试验,分别评估DCs的表型成熟、细胞因子表达和促进CD4~+T细胞增殖的能力。最后,在体外建立的DCs/Calu-3上皮细胞共培养模型和体内小鼠鼻腔灌注模型,应用流式细胞术和共聚焦显微镜等方法,探讨CpG诱导鼻腔黏膜下DCs跨上皮摄取H9N2 WIV并转运至引流淋巴结-颈淋巴结的能力,同时深入研究了DCs招募和树突形成的机制,也阐明了参与此过程的DCs亚型和捕获受体,以期为CpG ODN作为鼻腔黏膜免疫佐剂的研究和应用提供一定的理论依据。本研究内容分为以下三个部分:1、CpG ODN配合H9N2 WIV鼻腔免疫对小鼠呼吸道局部黏膜和全身系统免疫应答水平的影响本试验应用CpG ODN配合H9N2 WIV滴鼻免疫小鼠,通过检测鼻腔、气管和肺各呼吸道中IgA的特异性抗体水平;血清中IgG及其亚型抗体水平;中和HI水平;脾淋巴细胞的活化(CD69表达)、增殖和亚群变化的情况,评价小鼠局部黏膜和全身系统的免疫应答水平。结果发现:应用CpG ODN配合H9N2 WIV滴鼻免疫小鼠28天后,鼻腔、气管和肺涮洗液中IgA抗体水平、血清中IgG及其亚型抗体水平、中和HI水平均显著(p0.05)高于单独H9N2 WIV免疫后的水平。此外,脾淋巴细胞活化和增殖能力显著提升,CD3~+CD4~+T细胞亚群比例也有大幅上升。然而,单独应用H9N2 WIV免疫后,以上指标均未显著上调(p0.05).结果表明:CpG ODN作为鼻腔黏膜佐剂,配合H9N2 WIV后可以有效地提升小鼠呼吸道局部黏膜及全身的系统免疫应答水平。2、CpG ODN配合H9N2 WIV对小鼠树突状细胞活化和成熟的影响DCs是连接天然免疫和获得性免疫的重要的纽带。DCs活化和成熟直接影响着下游免疫应答水平。因此,本试验首先从小鼠骨髓中分离骨髓前体细胞,并应用GM-CSF和IL-4联合诱导前体细胞分化为DCs,并通过形态学、纯度和功能进行鉴定。随后,应用CpG ODN配合H9N2 WIV体外刺激已培养成功的DCs 24 h,收集DCs和上清液,分别检测DCs的成熟表型标志和细胞因子的表达。此外,另一部分收集的DCs与异种的CD4~+T细胞混合培养,检测CD4~+T细胞的增殖情况。结果显示:CpG ODN配合H9N2 WIV后,与空白对照组相比,显著上调CD40和CD80的表达;同时,促进IL-12p70和IL-10分泌;在DCs与CD4~+ T细胞的混合淋巴细胞反应试验中,CD4~+ T细胞显著增殖。结果表明:CpG ODN配合H9N2 WIV后可以有效地促进DCs活化和成熟,可能是引起机体较好免疫应答水平的重要机制。3、CpG ODN对鼻腔黏膜下DCs跨上皮摄取H9N2 WIV的影响CpG ODN是否能够有效协助H9N2 WIV跨鼻腔黏膜递送,目前仍不清楚。在本研究中,通过体外DCs/Calu-3共培养模型试验和体内小鼠鼻腔灌注试验,发现CpG ODN能有效协助H9N2 WIV招募DCs至鼻腔黏膜下区域,并伸出跨上皮树突捕获腔侧的病毒粒子。其中,CD103~+亚型DCs参与了以上过程,SIGN-R1是鼻腔黏膜DCs上重要的摄取H9N2 WIV的受体。鼻腔上皮细胞分泌的趋化因子CCL20在DCs招募和跨上皮树突形成的过程中发挥重要作用。摄取病毒的鼻腔黏膜DCs能够快速的向其引流淋巴结-颈淋巴结迁移并递呈抗原。此外,CpG ODN并没有影响上皮细胞自身的转运能力,即跨细胞途径和细胞旁通路。结果表明:CpG ODN配合H9N2 WIV后可以通过诱导黏膜下DCs形成跨上皮树突,进而有效地摄取H9N2 WIV,这可能是CpG ODN增强下游获得性免疫应答的新机制。
[Abstract]:Aivan Influenza (AI) has caused great economic loss to poultry industry in China and many countries in the world. Avian influenza is mainly divided into high pathogenic and low pathogenic avian influenza. Although the incidence and mortality of low pathogenic avian influenza are low, the host can transmit the virus as a carrier and provide the variant recombinant of influenza virus. The nasal cavity is one of the main entrance of the avian influenza virus into the body. The study shows that the nasal cavity immunity can establish an effective mucosal immune response in the local area and directly cut off the infection path of the virus. However, the inactivation of the inactivated influenza virus for nasal immunity can not be effectively promoted because of the obstruction of the nasal mucosa barrier. In recent years, CpG ODN (CpG Oligodeoxynucleotides) adjuvant has been widely concerned and studied the main mechanism of.CpG ODN because of its strong immune stimulation effect. It can target and promote the maturation of submucosal dendritic cells (Dendritic cells, DCs), and thus effectively enhance the mucosal and systemic immune response. In the presence of the mucosal barrier, it is not clear whether CpG ODN contributes to the transmission of the cross nasal mucosa of the avian influenza inactivated virus particles. Therefore, this study first confirms the local mucosal immunity and systemic immune response level of CpG ODN combined with H9N2 avian influenza inactivated virus (H9N2 WIV) inactivated virus (H9N2 WIV) mice. Again, in vitro, CpG ODN With the interaction of H9N2 WIV directly with mouse DCs (in vitro induced culture), flow cytometry, ELISA test and mixed lymphocyte reaction test were used to evaluate the phenotypic maturation of DCs, the expression of cytokines and the ability to promote the proliferation of CD4~+T cells. Finally, the co culture model of DCs/Calu-3 epithelial cells and the mice nose in vitro were established in vitro. Cavity perfusion model, flow cytometry and confocal microscopy were used to investigate the ability of CpG to induce DCs transepithelial uptake of H9N2 WIV under nasal mucosa and transport to lymph node and cervical lymph node. The mechanism of DCs recruitment and dendrite formation was studied, and the DCs subtype and capture receptor involved in the process were also clarified, so as to be CpG OD. N provides a certain theoretical basis for the research and application of nasal mucosal immune adjuvant. The contents of this study are divided into three parts: 1, the effect of CpG ODN combined with H9N2 WIV on the local mucosal and systemic immune response in the respiratory tract of mice. The experiment was conducted by CpG ODN combined with H9N2 WIV nose immunization in mice, through the detection of the nasal cavity, The specific antibody level of IgA in the trachea and the respiratory tract, the level of IgG and its subtype antibody in the serum, the level of neutralizing HI, the activation of the spleen lymphocyte (CD69 expression), the proliferation and subgroup changes, and evaluating the immune response level of the local mucosa and systemic system of the mice. The results showed that the use of CpG ODN combined with H9N2 WIV intranasal immune mice for 28 days After that, the level of IgA antibody in the nasal cavity, trachea and lung rinse, the level of IgG and its subtype antibody in the serum, and the level of the neutralizing HI (P0.05) were higher than that of the individual H9N2 WIV. In addition, the activation and proliferation of splenic lymphocytes increased significantly, and the CD3~+CD4~+T cell subsets were also significantly increased. However, H9N2 WIV immunization was used alone. The above indexes were not significantly up-regulated (P0.05). The results showed that CpG ODN, as a nasal mucosa adjuvant, combined with H9N2 WIV, could effectively enhance the systemic immune response level.2 in the local mucosa and whole body of the respiratory tract of mice. The effect of CpG ODN and H9N2 WIV on the activation and maturation of dendritic cells in mice was linked to natural and acquired immunity. The important link,.DCs activation and maturation, directly affects the level of the downstream immune response. Therefore, this experiment first isolated the bone marrow progenitor cells from the mouse bone marrow, and used GM-CSF and IL-4 to induce the differentiation of the precursor cells into DCs, and identified by morphology, purity and function. Then, CpG ODN combined with H9N2 WIV in vitro was used to stimulate the body. The successful DCs 24 h was cultured and DCs and supernatant were collected to detect the mature phenotypic markers of DCs and the expression of cytokine. In addition, the proliferation of CD4~+T cells was detected by the mixed culture of DCs and heterogeneous CD4~+T cells. The results showed that CpG ODN combined with H9N2 WIV, compared with the blank control group, the CD40 and the CD4~+T were significantly up-regulated. At the same time, it promotes the secretion of IL-12p70 and IL-10, and in the mixed lymphocyte reaction test of DCs and CD4~+ T cells, CD4~+ T cells proliferate significantly. The results show that CpG ODN and H9N2 WIV can effectively promote DCs activation and maturation, and may be an important mechanism to induce better immune response to the body. The effect of epithelial uptake of H9N2 WIV on CpG ODN is still unclear whether CpG ODN can effectively assist the H9N2 WIV delivery of nasal mucosa. In this study, the DCs/Calu-3 co culture model test in vitro and in vivo mouse nasal perfusion test found that CpG ODN could effectively assist H9N2 WIV to recruit DCs to submucosal region of the nasal cavity and extend the trans epithelial dendritic trap. The CD103~+ subtype DCs participates in the above process, and SIGN-R1 is an important receptor for the uptake of H9N2 WIV on the nasal mucosa DCs. The chemokine CCL20 secreted by the nasal epithelial cells plays an important role in the recruitment of DCs and the formation of the trans epithelial dendrites. Lymph nodes and cervical lymph nodes migrate and present antigen. In addition, CpG ODN does not affect the transport capacity of epithelial cells themselves, that is, cross cell pathway and paracellular pathway. The results show that CpG ODN can induce DCs to form transepithelial dendrites by inducing submucosal DCs and then effectively absorb H9N2 WIV after H9N2 WIV, which may be a CpG ODN enhanced downstream acquisition. A new mechanism for the response to the immune response.

【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S852.4

【相似文献】