CpGs对消化道黏膜树突状细胞跨上皮摄取灭活禽流感病毒的影响

发布时间：2018-06-24 09:08

本文选题：灭活H9N2禽流感病毒 + 树突状细胞　；参考：《南京农业大学》2015年博士论文

【摘要】：禽流感病毒的广泛传播不仅给世界各国的养禽业造成了巨大的经济损失,也加剧了新型流感爆发的风险。禽流感病毒除了可以通过呼吸道传播外,还可以在水禽的消化道内复制并通过粪便向外不断排毒。口服疫苗可以在消化道黏膜建立免疫防线,直接切断病毒的传播路径。尽管灭活禽流感病毒作为口服疫苗的候选抗原,较减毒活疫苗相比,具有较好的安全性,但是应用单独抗原进行免疫,免疫效果往往不理想。此外,在口服免疫途径中,肠道黏膜屏障是影响抗原跨上皮转运的一个不可忽略的关键因素,再加之灭活病毒的复制性丧失,导致黏膜下树突状细胞(Dendritic cells,DCs)捕获的抗原数量急剧减少,对启动下游抗原特异性免疫应答非常不利。CpGs是一种安全有效的黏膜免疫增强剂。本实验室的研究发现,CpGs配合灭活H9N2禽流感病毒(H9N2 whole inactivated influenza viruses,H9N2 WIV)口服免疫鸭可以显著增强局部黏膜和全身系统免疫应答水平。那么,CpGs除了可以促进免疫细胞的天然免疫外,是否在H9N2 WIV的跨上皮转运过程中发挥重要作用。因此,本研究首先确证了CpGs配合H9N2 WIV口服免疫后对小鼠局部黏膜和全身系统免疫应答水平的影响。其次,在体外试验中应用树突状细胞/肠上皮细胞(DC/Caco-2)共培养模型,在体内试验中应用肠道原位结扎灌注模型,深入剖析了CpGs在H9N2 WIV跨消化道上皮转运过程中发挥的作用。最后,在黏膜下DCs成功捕获抗原后,进一步探讨了DCs表型和功能成熟的情况。本研究内容具体分为以下三个部分:1、CpGs配合灭活H9N2禽流感病毒口服免疫对小鼠局部黏膜和全身系统免疫应答水平的影响本试验应用CpGs配合灭活H9N2禽流感病毒口服免疫小鼠,首先通过检测肠道、气管和肺涮洗液中特异性IgA抗体水平探讨CpGs配合H9N2 WIV口服免疫对局部黏膜免疫水平的影响。其次,通过检测血清特异性IgG及其亚型抗体水平、HI中和抗体水平、肠系膜淋巴结和脾脏淋巴细胞的活化和增殖情况、脾脏淋巴细胞亚群变化,探讨CpGs配合H9N2 WIV口服免疫对机体全身系统免疫应答水平的影响。结果显示:首免后28天,与单独H9N2 WIV免疫组相比,在CpGs配合H9N2 WIV口服免疫组中,肠道、气管和肺涮洗液中特异性IgA抗体水平显著提升;在全身系统免疫中,血清特异性IgG、IgG1、IgG2a/c抗体水平和HI水平显著提高,脾淋巴细胞中CD3+CD4+T细胞的比例明显上调;当抗原再次刺激时,肠系膜淋巴结和脾脏淋巴细胞活化标志CD69的表达显著上调、淋巴细胞的增殖能力显著增强。结果表明:CpGs配合H9N2 WIV口服免疫小鼠,可以显著提高局部消化道黏膜和全身系统免疫应答水平。2、CpGs对树突状细胞跨消化道黏膜上皮摄取灭活H9N2禽流感病毒能力的影响在体外试验中,建立DC/Caco-2共培养模型,在小鼠体内试验中,建立肠道原位结扎灌注模型,应用共聚焦显微镜和流式细胞技术,深入探讨了CpGs是否有助于H9N2 WIV的跨消化道上皮转运。结果显示:CpGs配合H9N2 WIV能够募集更多的DCs聚集在肠上皮下方,形成跨上皮树突(Transepithelial dendrites,TEDs),这些树突可以摄取肠腔中的H9N2 WIV和CpGs。这个过程并不依赖上皮的转胞吞方式和上皮屏障的破坏。此外,进一步发现CD103+和CD103-两种肠黏膜DCs亚型是这个过程的重要参与者。其中,摄取病毒粒子的CD103+ DCs可以在2 h内快速迁移至肠系膜淋巴结并参与抗原递呈。在回肠和空肠处,DCs募集和TEDs的形成更易发现,而在派尔氏斑(Peyer's patches,PPs)处,CpGs更有利于DCs向上皮下圆顶区聚集,而TEDs却非常少见。进一步研究发现,CpGs配合H9N2 WIV可以显著促进消化道上皮细胞表达趋化因子CCL20,进而募集更多的DCs在黏膜下聚集。CCL20在上皮细胞游离侧分布的方式,也为TEDs的形成提供了可能。结果表明:CpGs通过刺激肠上皮细胞分泌CCL20,募集DCs至肠黏膜下并形成TEDs,进而加强H9N2 WIV的跨上皮转运,这可能是引起下游有效的抗原特异性免疫应答的一个重要机制。3、CpGs配合灭活H9N2禽流感病毒对消化道黏膜树突状细胞成熟能力的影响黏膜下DCs摄取抗原后,将快速转变状态,由未成熟转变为成熟状态,以便下游的抗原递呈过程。因此,本研究中,在体外DC/Caco-2共培养模型中,通过检测黏膜下DCs的表型标志表达、细胞因子分泌和混合淋巴细胞反应能力,评价CpGs配合H9N2 WIV对黏膜下DCs成熟功能转变的情况。结果发现,在共培养系统的游离侧接种CpGs和H9N2 WIV并作用24 h后,与培养基对照组相比,显著上调DCs表型标志CD40、CD80、CD86和MHCⅡ的表达;同时,强烈刺激DCs分泌IL-10、IL-12p70和IL-23;混合淋巴细胞反应试验中,在DC:T=1:1和1:5两种细胞比例下,CD4+T细胞的增殖能力显著增强。结果表明:在体外DC/Caco-2共培养模型中,CpGs配合H9N2 WIV可以有效地增强黏膜下DCs的成熟能力,这对下游获得性免疫应答的启动具有重要作用。
[Abstract]:The spread of avian influenza virus has not only caused huge economic losses to poultry industry in the world, but also increased the risk of a new outbreak of influenza. In addition to the respiratory tract, the avian influenza virus can also be replicated in the digestive tract of waterfowl and can not be detoxicated through feces. In spite of the inactivation of the avian influenza virus as the candidate antigen of the oral vaccine, it has a better safety than the live attenuated vaccine, but the immune effect is often not ideal with the use of individual antigens. In addition, the intestinal mucosal barrier in the oral immune pathway affects the antigen span. A key factor that can not be ignored in skin transport, and the replication loss of inactivated virus, resulting in a sharp decrease in the number of antigens captured by the submucosal dendritic cells (Dendritic cells, DCs) and a very effective mucosal immune enhancement agent for the initiation of the specific immune response to the downstream antigen is a safe and effective mucosal immune enhancement agent. It was found that CpGs combined with inactivated H9N2 avian influenza virus (H9N2 whole inactivated influenza viruses, H9N2 WIV) oral immune ducks could significantly enhance the level of local mucosal and systemic immune responses. In addition, CpGs can play an important role in the trans epithelial transport of H9N2 WIV, in addition to promoting the natural immunity of immune cells. This study first confirmed the effect of CpGs combined with H9N2 WIV on the local mucosal and systemic immune response level in mice. Secondly, in vitro, the co culture model of dendritic cells / intestinal epithelial cells (DC/Caco-2) was used in the experiment, and the intestinal ligation perfusion model was used in the experiment in vivo, and CpGs was deeply analyzed in H9N2 WIV. Finally, after the submucosal DCs successfully captured the antigen, the DCs phenotype and functional maturity were further investigated. The content of this study was divided into three parts: 1, CpGs combined with inactivated H9N2 avian influenza virus oral immune response to local mucosal and systemic immune responses in rats The effect of CpGs combined with inactivated H9N2 avian influenza virus in mice was studied. First, the effects of CpGs and H9N2 WIV on the level of local mucosal immunity were investigated by detecting the specific IgA antibody level in the intestinal tract, trachea and lung rinse. Secondly, the serum specific IgG and its subtype antibody level, HI neutralizing antibody were detected. Levels, activation and proliferation of mesenteric lymph nodes and splenic lymphocytes, changes in lymphocyte subsets of the spleen and the effect of CpGs combined with H9N2 WIV oral immunization on systemic systemic immune response. The results showed that 28 days after the first immunization, compared with the single H9N2 WIV immunization group, the intestine and gas in the CpGs combined with the H9N2 WIV oral immune group. The level of specific IgA antibody in the tube and lung rinse fluid increased significantly. In systemic systemic immunization, the serum specific IgG, IgG1, IgG2a/c antibody level and HI level were significantly increased, and the proportion of CD3+CD4+T cells in the splenic lymphocyte was obviously up-regulated. When the antigen was stimulated again, the expression of the mesenteric lymph node and spleen lymphocyte activation marker CD69 expression was obvious. The proliferation ability of lymphocytes was significantly enhanced. The results showed that CpGs combined with H9N2 WIV in mice could significantly improve the local digestive tract mucosa and systemic immune response level.2. CpGs has an effect on the ability of dendritic cells to intake the ability to intake H9N2 avian influenza virus in the epithelium of the digestive tract and to establish DC/Caco in vitro. The -2 co culture model, in the mice test, established the intestinal ligation perfusion model, applied confocal microscopy and flow cytometry to explore whether CpGs helped H9N2 WIV translocation in the trans alimentary tract. The results showed that CpGs combined with H9N2 WIV could raise more DCs to accumulate below the intestinal epithelium and form trans epithelial dendrites (T). Ransepithelial dendrites, TEDs), these dendrites can absorb the H9N2 WIV and CpGs. in the intestinal cavity, which does not depend on the epitheliocytosis and the destruction of the epithelial barrier. Furthermore, the DCs subtypes of the two intestinal mucosa of the CD103+ and CD103- are an important participant in this process. Among them, CD103+ DCs for the uptake of viral particles can be 2. H is rapidly migrated to the mesenteric lymph nodes and participates in antigen presentation. In the ileum and jejunum, DCs recruitment and the formation of TEDs are more easily found, while in Peyer's patches (PPs), CpGs is more conducive to the aggregation of DCs to the upper subcutaneous dome, but TEDs is very rare. Further studies have found that CpGs with H9N2 WIV can significantly promote digestion. The expression of chemokine CCL20 in the epithelial cells and further recruitment of more DCs in submucosal aggregation of.CCL20 in the free side of epithelial cells also provides a possibility for the formation of TEDs. The results show that CpGs enhances the secretion of CCL20 by stimulating the intestinal epithelial cells, raising DCs to the intestinal mucosa and forming TEDs, and thus enhancing the trans epithelial transport of H9N2 WIV. It may be an important mechanism to induce the effective antigen specific immune response in the downstream,.3, and CpGs combined with the inactivation of H9N2 avian influenza virus to the maturation of the dendritic cells in the digestive tract. After the submucosal DCs uptake antigen, it will change rapidly from immature to mature, so that the downstream antigen presentation process. In the study, in the in vitro DC/Caco-2 co culture model, by detecting the phenotype markers expression of submucosal DCs, cytokine secretion and mixed lymphocyte reaction ability, the changes of CpGs and H9N2 WIV on submucosal DCs maturation were evaluated. The results showed that the free side of the co culture system was inoculated with CpGs and H9N2 WIV and the action of 24 h. Compared with the control group, the expression of DCs phenotype marked CD40, CD80, CD86 and MHC II, and DCs secretion of IL-10, IL-12p70 and IL-23; in mixed lymphocyte reaction test, the proliferation ability of CD4+T cells increased significantly under the ratio of DC:T=1:1 and 1:5 two cells. H9N2 WIV can effectively enhance the maturation ability of submucosal DCs, which plays an important role in the initiation of downstream 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】：S852.65

【相似文献】