【作者】 张媛媛；

【导师】 黄克和； 陈瑾；

【作者基本信息】 南京农业大学 ， 兽医硕士（专业学位）， 2022， 硕士

【摘要】 树突状细胞(Dendritic Cell,DCs)作为专职抗原递呈细胞(Antigen Presenting Cell,APC),是目前公认递呈能力最强的细胞,是先天免疫和获得性免疫的桥梁。活化的DCs由注射部位迁移到引流淋巴结,进而诱导幼稚性T淋巴细胞分化为不同类型的效应T细胞,对于启动和调节CD4~+和CD8~+T细胞免疫反应至关重要,对机体产生获得性免疫应答起关键作用。细胞免疫是由T淋巴细胞介导的免疫。CD4~+T淋巴细胞介导的分泌IFN-γ为主的Th1型免疫应答和分泌IL-4为主的Th2型免疫应答在抗感染免疫中发挥重要作用;CD8~+T淋巴细胞也称细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL),与自然杀伤细胞构成机体抗病毒、抗肿瘤免疫的重要防线;CD8~+T细胞还可以通过表达分泌诸如TNF-α、IFN-γ等细胞因子来间接杀死靶细胞。CD8~+T淋巴细胞不仅可以有效杀灭胞内感染细胞,而且在易变异毒株间因交叉保护不足的病毒疫苗研制上,CTL应答可以提供高水平的交叉保护。本课题首先体外诱导培养C57BL/6小鼠DCs,评价CVC1302促进DCs活化、抗原交叉递呈能力、分泌细胞因子水平及胞内环境变化等,其次体内试验评价CVC1302配伍抗原免疫小鼠后促进抗体、抗体分型、CD8~+T细胞活化及特异性CTL能力,了解CVC1302对细胞免疫,特别是CD8~+T细胞免疫的提升,进而阐释其作用机制,为高效灭活疫苗的研制提供理论支撑。论文研究内容分为以下两部分:试验一CVC1302促进DCs活化及交叉递呈能力本实验首先分离6～8周龄C57BL/6小鼠骨髓源细胞,体外培养获得DCs,利用无内毒素OVA抗原及免疫增强剂CVC1302进行体外刺激,分别设CVC1302+OVA组、单独OVA组和空白对照组,通过流式细胞术检测DCs活化比率及其表面共刺激分子CD40、CD80、CD86及MHC-I的表达量;检测刺激24 h后,细胞上清中细胞因子(TNF-α、IFN-γ、IL-12p70)含量;利用活性氧(ROS)试剂盒检测刺激1、3 h后DCs胞内ROS变化趋势;CVC1302+OVA-FITC和OVA-FITC分别体外刺激后40、60 min,利用SIM超高分辨显微镜观察DCs胞内OVA-FITC的数量、存在时间及与溶酶体定位的变化情况;利用负载抗原DCs与B3Z细胞共同培养,检测B3Z细胞整体活化情况。结果显示:CVC1302+OVA组与单纯OVA组相比,DCs活化率显著提升(p<0.001),表面成熟分子CD40、CD80、CD86及MHC-I的表达量显著提升(p<0.001、p<0.01、p<0.01,p<0.001),DCs分泌细胞因子TNF-α、IFN-γ、IL-12p70水平显著提升(p<0.0001、p<0.01、p<0.0001);CVC1302提升DCs吞噬抗原能力、留存时间及胞内ROS水平,且抗原与溶酶体共定位减少;CVC1302+OVA组与单独OVA组相比,DCs活化B3Z细胞量显著提升(p<0.0001)。本实验成功建立了利用Flt3-L对C57BL/6小鼠骨髓源树突细胞体外培养及鉴定,证明CVC1302可显著提高体外培养DCs的活化率及相关表面分子表达量,并促进DCs对抗原的吞噬和交叉递呈的发生,为后续免疫增强剂CVC1302介导细胞免疫的机制研究,奠定了试验基础。试验二CVC1302对机体特异性免疫的影响为探究免疫增强剂CVC1302在小鼠体内对细胞免疫的影响,本实验利用模式抗原OVA,制备疫苗免疫C57BL/6小鼠,通过ELISA检测特异性血清抗体水平,利用流式细胞术分析引流淋巴结DCs数目和CD8~+T细胞增殖水平并评价体内CTL杀伤效率。通过免疫6～8周龄C57BL/6小鼠,分别设CVC1302+OVA组、单独OVA组和空白对照组;眼眶采血并分离血清,利用ELISA检测免疫28、60、90及120d的小鼠血清中特异性抗体及亚型Ig G1和Ig G2a的效价;利用流式细胞术检测免疫后1、3、5、7d小鼠引流淋巴结中DCs数目和CD8~+T细胞活化水平,同时检测免疫7d后小鼠体内特异性CTL杀伤效率,结果显示:CVC1302+OVA组与单纯OVA组相比,免疫小鼠28、60、90及120d后,血清抗体Ig G效价水平显著提升,差异极显著(p<0.001、p<0.01、p<0.05、p<0.05),抗体亚型Ig G1差异不显著(p>0.05),但是Ig G2a差异极显著(p<0.0001、p<0.0001、p<0.0001、p<0.01);CVC1302+OVA组与单纯OVA组对比,免疫后1、3、5、7d,小鼠引流淋巴结中DCs招募数量和CD8~+T细胞数量极显著提升(p<0.0001);体内CTL杀伤水平显著升高(p<0.01)。本实验成功验证CVC1302有利于体内特异性抗体水平显著提升,确认CVC1302对小鼠引流淋巴结募集DCs和活化CD8~+T细胞有明显促进作用,对机体发挥CTL反应具有显著提升影响,为后续免疫增强剂CVC1302在不同血清型疫苗上的交叉免疫保护作用提供研究数据。更多还原

【Abstract】 Dendritic cells(DCs),as the professional antigen presenting cells(APC),are recognized as the cells with the strongest presenting ability.DCs are a bridge between innate immunity and acquired immunity.Activated DCs migrate from the injection site to draining lymph nodes,then induce immature T lymphocytes to differentiate into different types of effector cells,which is crucial to initiating and regulating CD4~+and CD8~+T cell immune response,and plays a key role in the acquired immunity.Cellular immunity is mediated by T lymphocytes.CD4~+T lymphocytes,including Ig G1 subtype induced humoral immune antibodies and Ig G2a subtypes induced cellular immune responses and cytokines,also play an important role in anti-infection immunity.CD8~+T lymphocytes,also known as cytotoxic T lymphocytes(CTL),play a decisive role in the elimination of virus infected cells;CD8~+T cells can also release cytokines such as TNF-α、IFN-γand other cytokines to indirectly kill target cells.CD8~+T lymphocytes can not only effectively kill intracellular infected cells,but also provide a high level of cross protection against variant virus strain.In this study,firstly,we analyze the activation and antigen cross presentation of DCs,the level of cytokines secreted by DCs and the changes of DCs’intracellular environment,when DCs co-cultured with CVC1302 in vitro.Then,we evaluate the levels of antibody,subtype antibodies and the CD8~+T cell activation,as well as the specific CTL ability of mice immunized with OVA adjuvanted with CVC1302.The study aims to eclucidate the mechanism of CVC1302 in improving the cellular immunity in order to provide the theoretical support for the development of highly effective inactivated vaccine.The research contents of this paper are divided into the following two parts:Experiment I CVC1302 promotes DCs activation and cross-presentation in vitroIn order to explore the ability of CVC1302 to activate DCs and promote antigen cross presentation,an in vitro cultural technique of DCs from C57BL/6 mice was established.After stimulation with related antigens,DCs were used to evaluate the activation rate and surface mature molecular expression,antigen phagocytosis and existent time by flow cytometry,as well as the ability of DCs’antigen cross presentation to B3Z cells.The bone marrow cells from six-to-eight week old C57BL/6 mice were isolated and cultured,then,the activation rate of DCs was identified by flow cytometry.Endofree OVA antigen and immunopotentiator CVC1302 were used to stimulate DC in vitro.CVC1302+OVA group,single OVA group and blank control group were set up respectively.The activation ratio of DCs and the expression of surface costimulatory molecules CD40,CD80,CD86 and MHC-I were detected by flow cytometry;After 24 hours’stimulation,the levels of cytokines in cell supernatant were detected(TNF-α、IFN-γ、IL-12p70)by using commercial ELISA kits;reactive oxygen species(ROS)kit was used to detect the change trend of intracellular ROS in DCs after stimulation within 1 or 3 h;At 40 or 60 min post CVC1302+OVA-FITC or OVA-FITC stimulation in vitro,the number,existent time and lysosomal co-location of OVA-FITC in DCs were observed by SIM ultra-high resolution microscope;The researches showed that the activation rate of DCs in CVC1302+OVA group was significantly higher than that in OVA group(p<0.001),the expression of surface mature molecules CD40,CD80,CD86 and MHC-I was significantly higher(p<0.001,p<0.01,p<0.01,p<0.001),and the levels of DCs’secreted cytokines TNF-α、IFN-γ、IL-12p70 increased significantly(p<0.0001,p<0.01,p<0.0001);CVC1302increased the number of phagocytic antigens,existent time and intracellular ROS level of DCs,and decreased the protition of antigen co-located with lysosomes;The amount of B3Z cells activated by DCs in CVC1302+OVA group was significantly higher than that in OVA group alone(p<0.0001).In this study,we successfully seprated and cultured DCs from C57BL/6 mouse in vitro,stimulated with Flt3-L.On this basis,we found that CVC1302could significantly improve the activation rate and the expression of related surface molecules of DCs in vitro and promote the phagocytosis and cross-presentiation of antigens,which laid an experimental foundation for the subsequent study on explanation the mechanism of the immunopotentiator CVC1302 in inducing the cellular immunity.ExperimentⅡEffects of CVC1302 on body specific immunityIn order to evaluate the effect of immunopotentiator CVC1302 on specific humoral and cellular immunity in mice,C57BL/6 mice were immunized,the specific serum antibody level was detected by ELISA,the proliferation of DCs and CD8~+T cells in draining lymph nodes were analyzed by flow cytometry,as well as the killing efficiency of CTL.Six-to-eight weeks old C57BL/6 mice were randomly seprated into three groups,name as CVC1302+OVA group,OVA group or blank control group,respectively.Blood samples were collected at 28,60,90 and 120d post immunization,the levels of specific antibody and isotype antibodies(Ig G1 and Ig G2a)were detected by ELISA.Flow cytometry was used to detect the recruiment of DCs and the activation of CD8~+T cells in the draining lymph nodes of mice at 1,3,5 and 7d after immunization,as well as the specific CTL killing efficiency of mice at 7d after immunization.Results:compared with the simple OVA group,the serum antibody Ig G titer level of CVC1302+OVA group significantly enhanced at 28,60,90 and 120d after immunization(p<0.001,p<0.01,p<0.05 and p<0.05).There was no significant difference in antibody subtype Ig G1(p>0.05),but the difference on the level of Ig G2a was very significant(p<0.0001,p<0.0001,p<0.0001,p<0.01).Group CVC1302+OVA,compared with group OVA,the numbers of recruited DCs and activated CD8+T cells in draining lymph nodes increased significantly.There was a significant increase in the level of CTL killing in vivo(p<0.01).The results showed that CVC1302 could not only significantly increase the specific antibody level in vivo,but also significantly promote the recruitment of DCs and activation of CD8~+T cells,as well as the ability of CTL in mouse draining lymph nodes.Overall,the study provide a research platform for the application of subsequent immunopotentiator CVC1302 on different serotype vaccines to induce cross immune response.更多还原