小鼠树突状细胞提呈口蹄疫病毒蛋白质抗原的机制研究

发布时间：2018-07-02 23:51

本文选题：树突状细胞 + 口蹄疫病毒　；参考：《河北医科大学》2008年博士论文

【摘要】： 目的:口蹄疫(foot-and-mouth disease,FMD )是由口蹄疫病毒(foot-and-mouth disease virus, FMDV)引起的牛、羊、猪等偶蹄动物的一种急性、高度接触性、发热性传染病。FMDV也可感染人类,并表现出与动物类似的临床症状。因此,被世界卫生组织(WHO)把口蹄疫定为人兽共患传染病。虽然该病已经存在了四百多年,但尚不清楚机体对FMDV的免疫应答机制。树突状细胞(dendritic cells,DC)不仅是动物机体的哨位细胞(sentinel cell),而且还是机体适应性免疫应答的启动者,特别是DC还能够交叉提呈非复制性抗原(例如灭活的病毒等),故被称为机体最强大的抗原提呈细胞,但目前尚不清楚DC是否参与加工、提呈FMDV抗原。由于国家严格规定从事口蹄疫活病毒研究必须在生物安全三级实验室以上的环境条件下进行,因此,本研究以灭活FMDV疫苗为抗原材料,通过观察口服灭活FMDV疫苗后咽部引流淋巴结内树突状细胞的分布变化,明确DC是否作为FMDV的抗原提呈细胞参与免疫应答,并进一步研究DC提呈FMDV抗原的机制。近年来,FMDV感染模型已经在BALB/c小鼠成功复制,因此,如果试验结果证实了DC是灭活FMDV的抗原提呈细胞,将以BALB/c小鼠为试验对象,通过体内外试验,进一步探讨小鼠树突状细胞向CD4+T细胞和CD8+T细胞提呈灭活FMDV抗原的机制。适应性免疫包括细胞免疫和体液免疫两部分。众所周知,如果没有细胞免疫应答的适当启动,特别是由CD4+T细胞介导的免疫应答,体液免疫应答就不可能得到建立,而且由CD8+T细胞介导的细胞免疫也是机体抵御病毒感染最有效的机制之一,所以,本研究选用CD4+T细胞和CD8+T细胞作为接受DC提呈抗原的靶细胞。同时,CD4+T细胞和CD8+T细胞的活化也可作为反映MHC-II类分子和MHC-I类分子途径提呈抗原的指标。方法:FMDV主要经消化道、呼吸道和受损伤的皮肤等途径感染动物,并可在咽部大量繁殖,无论是自然感染动物,还是疫苗接种动物。因此,咽部可能在机体抗FMDV感染免疫中发挥特殊重要作用。根据黏膜免疫学理论,咽部引流淋巴结就是咽部黏膜免疫应答的诱导区,而咽部黏膜就是效应区。 1.DC与FMDV关系的确定:为探明咽部DC是否参与灭活FMDV抗原的加工与提呈,研究者模拟自然感染途径给BALB/c小鼠口服接种灭活FMDV疫苗,然后,在接种疫苗后不同时间点摘取咽部引流淋巴结(用墨汁追踪法证实小鼠咽部引流淋巴结为颈浅淋巴结),按常规进行固定、脱水、透明和包埋,制备6-8μm厚石蜡组织切片,用兔抗牛S-100多克隆抗体对淋巴结进行免疫组织化学染色,观察颈浅淋巴结内DC的分布,根据DC在颈浅淋巴结内的数量变化与分布变化判断其是否参与FMDV抗原的提呈。 2.口服灭活FMDV疫苗诱导的细胞免疫应答:将36只8周龄BALB/c小鼠随机分为6组,每组6只。1组、2组、3组、4组、5组小鼠口服灭活Asia I-O型双价灭活疫苗(100μl/只),并分别于接种疫苗后24h、48h、72h、96h、120h在无菌条件下收集外周血,制备血清。第6组小鼠口服灭菌PBS(pH7.2)缓冲液(100μl/只)作为对照。以血清IFN-γ水平为指标(反映机体的Th1应答和CD8+T细胞应答),用ELISA法检测小鼠对灭活FMDV疫苗的细胞免疫应答。 3. BALB/c小鼠单核细胞源树突状细胞(monocyte-derived dendritic cells, MoDC)的制备:按常规方法进行,并用免疫细胞化学法和流式细胞术对其进行鉴定。 4. MoDC对灭活FMDV抗原的泛素化作用:将灭活FMDV疫苗负载于MoDC,并在RPMI 1640培养基中进行细胞培养,然后在不同时间点将MoDC裂解,以裂解液为材料进行SDS-PAGE,转膜后用western blotting法检测MoDC内的泛素化蛋白以及泛素化作用出现的时间。 5.负载灭活FMDV的MoDC对淋巴结T细胞免疫应答的启动作用:将负载了灭活FMDV的MoDC与淋巴结CD4+T细胞和CD8+T细胞在RPMI 1640培养基中进行共培养。用抗CD4抗体或抗CD8抗体分别阻断CD4+T细胞或CD8+T细胞的活化,分别在第9h、12h、24h、36h、48h收集上清液,用ELISA检测IFN-γ的含量。 6. MoDC提呈灭活FMDV抗原的机制:分别用溶酶体抑制剂和蛋白酶体抑制剂处理MoDC,2h后将灭活FMDV负载于MoDC,随即与CD4+T细胞或CD8+T细胞共培养,分别在第9h、12h、24h、48h收集上清液,用ELISA检测其IFN-γ的含量。结果:1. DC与FMDV关系的确定:小鼠颈浅淋巴结内DC的数量在口服FMDV灭活疫苗24h后开始增多,散在分布于皮质区内。48h后小鼠颈浅淋巴结内DC的数量明显增多,并且主要分布于副皮质区。在72h后,咽部DC向颈浅淋巴结副皮质区的迁移达到顶峰,依然聚集在副皮质区。至96h,颈浅淋巴结内DC的数量急剧下降,在皮质区和副皮质区内只有少量DC呈现散在分布状,此即树突状细胞疲劳(dendritic cell exhaustion)。然而,120h后,颈浅淋巴结内DC的数量再次增多,且主要分布于皮质区。而PBS对照组未见类似改变。这说明DC就是FMDV的抗原提呈细胞。 2.口服灭活FMDV疫苗诱导的细胞免疫应答:接种疫苗24h后,小鼠血清内的IFN-γ含量开始升高(10.1568±0.4689),但与PBS对照组相(10.0512±0.0684)比差异不显著(P0.05),而48h后,接种疫苗组小鼠血清内的IFN-γ含量明显升高(10.7161±0.2199),与PBS对照组(10.0748±0.0571)相比,差异极显著(P0.01)。在接种72h后,接种疫苗组小鼠血清内的IFN-γ含量达到最高峰(11.6784±0.4590),与PBS对照组(10.1249±0.1498)相比,差异极显著(P0.01)。96h后,IFN-γ含量开始下降(10.9839±0.3633),与PBS对照组(10.0933±0.0954)相比,差异显著(P0.05),到120h时,试验组(10.5285±1.3903)与PBS对照组相(10.0534±0.1543)比差异不显著(P0.05)。这表明口服灭活FMDV疫苗所诱导的细胞免疫应答与DC向引流淋巴结内的迁移模式相一致。 3. BALB/c小鼠MoDC的制备:免疫细胞化学试验和流式细胞术检测均显示,本试验所制备的MoDC纯度大于99%。 4. MoDC对灭活FMDV抗原的泛素化作用:所有负载灭活FMDV MoDC中均发生了FMDV抗原的泛素化现象,而未负载灭活FMDV的MoDC中泛素化蛋白质检测则呈现阴性反应。在负载灭活FMDV疫苗0.5h后,MoDC内就出现了泛素化FMDV蛋白,分子量大约为75kD;3h后,泛素化蛋白含量稍有增加,但从不同时间点来看,泛素化蛋白含量变化趋势不明显。本试验所使用的两种单克隆抗体—鼠抗多链泛素化蛋白质单克隆抗体和鼠抗单链泛素化蛋白质单克隆抗体(克隆号分别为FK1和FK2)均在western blot试验中呈现阳性反应,说明MoDC以两种不同的方式对灭活FMDV蛋白质抗原进行泛素化。 5. MoDC提呈灭活FMDV抗原的机制:在灭活FMDV负载MoDC与CD4+T细胞共培养9h即有大量IFN-γ产生,而与FMDV负载MoDC共培养的CD8+T细胞在受到抗原刺激后24h才大量释放IFN-γ。用溶酶体抑制剂或蛋白酶体抑制剂分别处理MoDC,2h后再分别与CD4+T细胞或CD8+T细胞共培养,按前述方法检测上清液中IFN-γ的含量,结果发现,两种抑制剂均在共培养后9h显著抑制CD4+T细胞产生IFN-γ。值得注意的是,溶酶体抑制剂显著抑制灭活FMDV负载DC刺激CD8+T细胞产生IFN-γ的能力,而蛋白酶体抑制剂却促进CD8+T细胞产生IFN-γ,但是,同时用两种抑制剂处理DC,CD8+T细胞产生IFN-γ的能力又显著下降。这些复杂现象提示MoDC主要以交叉提呈抗原的方式激活淋巴结T细胞,并以产生IFN-γ为特征。但是,CD8+T细胞产生IFN-γ的时间明显晚于CD4+T细胞,并且IFN-γ释放也呈现多样性。结论:1.咽部DC是灭活FMDV的抗原提呈细胞,但在DC捕获灭活FMDV抗原后的迁移过程中表现出“疲劳”特征(Dendritic cell exhaustion)。 2.小鼠口服接种灭活FMDV疫苗可以诱导细胞免疫应答,并以产生IFN-γ为特征。 3.灭活FMDV抗原被DC捕获后发生了两种不同的泛素化现象,即多链泛素化和单链泛素化。但泛素化FMDV蛋白质抗原主要被FK2抗体标记,而FK1抗体标记的较少。由于FK1只特异性地结合多链泛素化蛋白质,FK2则同时结合单链泛素化蛋白质和多链泛素化蛋白质,并且二者都不与游离的泛素结合。所以,灭活FMDV蛋白质抗原主要在DC内被多链泛素化。 4.泛素化的FMDV蛋白质抗原既可被蛋白酶体降解,也可在溶酶体内被加工处理,所产生的抗原肽主要以交叉提呈的方式提呈给CD4+T细胞或CD8+T细胞。CD4+T细胞只能识别装载着抗原表位的MHC-II类分子,所以,溶酶体抑制剂造成的IFN-γ含量显著下降清楚地表明,DC能够以溶酶体-MHC-II类分子途径按常规提呈FMDV抗原。而蛋白酶体抑制剂对CD4+T细胞分泌IFN-γ的强大抑制作用则提示,DC内还可能存在以蛋白酶体-MHC-II类分子途径交叉提呈FMDV抗原的机制。并且CD4+T细胞被抗原肽激活后主要分化为Th1细胞亚类,分泌高水平的IFN-γ。从上述结果不难看出,FMDV负载的DC则主要以“内吞小体-溶酶体-MHC-I类分子途径”交叉提呈抗原肽给CD8+T细胞,从而启动CTL应答。与DC迁移疲劳现象一致,CD8+T细胞产生IFN-γ的能力在被活化后48h显著下降,也呈现出向抑制的应答疲劳状态。
[Abstract]:Objective: foot-and-mouth disease (FMD) is an acute, highly contagious, infectious disease of cattle, sheep, pigs and other hoofed animals caused by foot-and-mouth disease virus (foot-and-mouth disease virus, FMDV), and the febrile infectious disease.FMDV can also infect humans and show similar clinical symptoms. Therefore, the WHO (WHO) has a foot-and-mouth disease. Although it has been in existence for more than 400 years, it is not clear that the body's immune response to FMDV is not clear. Dendritic cells (DC) is not only the post cell (sentinel cell) of animal body (sentinel cell), but also the promoter of the adaptive immune response to the organism, especially the DC can also be non replicating. Sex antigen, such as inactivated virus, is known as the most powerful antigen presenting cell of the body, but it is not clear whether DC is involved in processing and presenting FMDV antigen. Because the state strictly stipulates that the study of foot-and-mouth disease virus must be carried out under the environmental conditions above the biological safety level 3 Laboratory, therefore, this study is to inactivate the FMDV epidemic. The vaccine was an antigen material. By observing the distribution of dendritic cells in the pharynx drainage lymph node after oral inactivation of FMDV vaccine, it is clear whether DC is an antigen presenting cell of FMDV and participates in the immune response, and the mechanism of FMDV antigen presentation by DC has been further studied. In recent years, the FMDV infection model has been successfully replicated in BALB/c mice. Therefore, if the test has been done, if the test has been done, the test of the FMDV infection model has been successfully replicated. The test results confirmed that DC is an antigen presenting cell for inactivating FMDV. It will take BALB/c mice as the test object. Through the experiment in vitro and in vivo, the mechanism of inactivating FMDV antigen by mouse dendritic cells to CD4+T and CD8+T cells is further explored. Adaptive immunity includes two parts of cell immunity and humoral immunity. The appropriate start of the epidemic, especially the immune response mediated by CD4+T cells, can not be established in the humoral immune response, and the cellular immunity mediated by CD8+T cells is also one of the most effective mechanisms for resisting the virus infection. Therefore, this study selects CD4+T cells and CD8+T cells as the target cells for receiving DC antigen presenting antigen. At the same time, the activation of CD4+T cells and CD8+T cells can also serve as indicators of antigen presentation of MHC-II molecules and MHC-I molecular pathways.
Methods: FMDV mainly infect animals through the digestive tract, respiratory tract and damaged skin, and can proliferate in the pharynx, whether it is a natural infection or a vaccine. Therefore, the pharynx may play a special role in the immunity of the body against FMDV infection. According to the theory of mucosal immunology, the pharynx drainage lymph node is the pharynx. Mucosal immune response is induced, while pharyngeal mucosa is the effector area.
The determination of the relationship between 1.DC and FMDV: to determine whether the pharyngeal DC was involved in the processing and presentation of the inactivated FMDV antigen, the researchers simulated the natural infection route to the BALB/c mice by oral inactivation of the FMDV vaccine. Then, the pharynx drainage lymph nodes were extracted at different time points after the inoculation (with the ink tracing method confirmed that the throat drainage lymph nodes of the mice were superficial to the neck. " The 6-8 m thick paraffin tissue sections were prepared by routine fixation, dehydration, transparency and embedding. The lymph nodes were stained with Rabbit anti bovine S-100 polyclonal antibody to observe the distribution of DC in the superficial cervical lymph nodes. According to the changes and distribution changes of the number of DC in the superficial cervical lymph nodes, they were judged to be involved in the presentation of the FMDV antigen.
2. the cellular immune response induced by oral inactivated FMDV vaccine: 36 8 week old BALB/c mice were randomly divided into 6 groups, each group of 6.1 groups, 2 groups, 3 groups, 4 groups, and 5 groups of mice inactivated Asia I-O bivalent inactivated vaccine (100 mu l/), and after inoculation, 24h, 48h, 72h, 96h, 120h were collected under aseptic conditions to prepare serum and sixth mice. The oral sterilization PBS (pH7.2) buffer solution (100 mu l/) was used as control. The serum IFN- gamma level was used as an index (reflecting the body's Th1 response and CD8+T cell response), and the cellular immune response of mice to inactivated FMDV vaccine was detected by ELISA method.
3. BALB/c mouse monocyte derived dendritic cells (monocyte-derived dendritic cells, MoDC) were prepared by routine methods and identified by immunocytochemistry and flow cytometry.
4. MoDC to inactivate FMDV antigen ubiquitination: inactivated FMDV vaccine loaded with MoDC, and cell culture in the RPMI 1640 medium, and then cracking MoDC at different time points, using lysate as the material for SDS-PAGE, Western blotting method for the detection of ubiquitination protein in MoDC and the time of ubiquitination in MoDC.
5. load inactivated FMDV MoDC on the immune response of T cells in lymph nodes: co culture MoDC with inactivated FMDV and lymph node CD4+T cells and CD8+T cells in RPMI 1640 medium. The activation of CD4+T cells or CD8+T cells was blocked with anti CD4 or anti CD8 antibodies. The content of IFN- gamma was detected by ELISA.
6. MoDC proposed the mechanism of inactivating FMDV antigen: MoDC was treated with lysosome inhibitor and proteasome inhibitor respectively. After 2h, the inactivated FMDV was loaded on MoDC, then CD4+T cells or CD8+T cells were co cultured, and the supernatant was collected in 9h, 12h, 24h, 48h, respectively.
Results: the determination of the relationship between 1. DC and FMDV: the number of DC in the superficial cervical lymph nodes in mice began to increase after the oral FMDV inactivated vaccine 24h. The number of DC in the superficial cervical lymph nodes scattered in the cortical region was significantly increased and mainly distributed in the paracortical area. After 72h, the migration of the pharynx from the pharynx to the superficial lymph node paracortical area reached the peak. The number of DC in the superficial cervical lymph nodes decreased dramatically in the superficial cervical lymph nodes, and only a small amount of DC in the cortical and paracitical areas showed scattered distribution in the cortex and paracitical areas, that is, dendritic cell fatigue (dendritic cell exhaustion). However, the number of DC in the superficial cervical lymph nodes increased again after 120h, and was mainly distributed in the cortical area. The PBS control group was mainly distributed in the cortical region. No similar changes were observed. This indicates that DC is the antigen presenting cell of FMDV.
2. the cellular immune response induced by oral inactivated FMDV vaccine: after vaccination 24h, the content of IFN- gamma in the serum of mice began to increase (10.1568 + 0.4689), but (10.0512 + 0.0684) compared with the PBS control group (P0.05), but after 48h, the content of IFN- gamma in the serum of the vaccinated mice increased significantly (10.7161 + 0.2199), and the control group of PBS (10.) (10.) The difference was very significant (P0.01). After inoculation of 72h, the content of IFN- gamma in the serum of vaccinated mice reached the highest peak (11.6784 + 0.4590). Compared with the PBS control group (10.1249 + 0.1498), the difference was very significant (P0.01).96h, the content of IFN- gamma decreased (10.9839 + 0.3633), and the difference was significant compared with that of the PBS control group (10.0933 + 0.0954). (P0.05), at 120h, the test group (10.5285 + 1.3903) and the PBS control group (10.0534 + 0.1543) had no significant difference (P0.05). This showed that the cellular immune response induced by the oral inactivated FMDV vaccine was in accordance with the migration patterns of DC in the drainage lymph nodes.
3. the preparation of MoDC in BALB/c mice: immunocytochemistry and flow cytometry showed that the purity of MoDC prepared in this experiment was greater than 99%.
4. MoDC's ubiquitination of inactivated FMDV antigen: the ubiquitination of FMDV antigen occurred in all load inactivated FMDV MoDC, while the ubiquitinated protein detection in MoDC without inactivated FMDV showed negative reaction. The FMDV protein appeared in MoDC after FMDV vaccine 0.5h, and the molecular weight was about 75kD. The content of protein-protein was slightly increased, but the change trend of ubiquitinated protein content was not obvious at different time points. The two monoclonal antibodies used in this experiment - mouse anti - Multi Chain ubiquitinated protein monoclonal antibody and mouse anti - single chain ubiquitinated protein monoclonal antibody (clone number FK1 and FK2, respectively) were presented in the Western blot test The positive reaction indicates that MoDC is ubiquitin to inactivate FMDV protein antigen in two different ways.
5. MoDC presented a mechanism for inactivating FMDV antigen: a large number of IFN- gamma produced by the co culture of 9h in the inactivated FMDV load MoDC and CD4+T cells, while the CD8+T cells co cultured with FMDV loaded MoDC only released IFN- gamma in large quantities after being stimulated by the antigen. 8+T cells were co cultured and detected the content of IFN- gamma in the supernatant according to the previous methods. The results showed that the two inhibitors all inhibited the production of IFN- gamma in CD4+T cells after co culture. It is worth noting that the lysosome inhibitor significantly inhibited the ability of the inactivated FMDV load DC to stimulate CD8+T cells to produce IFN- gamma, while the proteasome inhibitor promoted CD8+T to be fine. IFN- gamma is produced by the cell, but the ability to produce IFN- gamma by two inhibitors and the production of IFN- gamma in CD8+T cells is significantly decreased. These complex phenomena suggest that MoDC mainly activates the lymphoid T cells with a cross presenting antigen, and is characterized by the production of IFN- gamma. However, the time of producing IFN- gamma in CD8+T cells is significantly later than CD4+T cells and IFN- gamma release. It also showed diversity. Conclusion: 1. DC of the pharynx is an antigen presenting cell for inactivating FMDV, but it shows "fatigue" (Dendritic cell exhaustion) during the migration of the inactivated FMDV antigen by DC.
2. mice inoculated with inactivated FMDV vaccine can induce cellular immune response and produce IFN- gamma.
3. inactivated FMDV antigen was captured by DC after two different ubiquitination, namely, Multi Chain ubiquitination and single chain ubiquitination. But ubiquitination FMDV protein antigen is mainly marked by FK2 antibody, while FK1 antibody is less marked. Because FK1 only specifically combines multi chain ubiquitination protein, FK2 combines single chain ubiquitination protein and multi chain simultaneously. Ubiquitin protein, and the two do not bind to free ubiquitin, so the inactivated FMDV protein antigen is mainly ubiquitin ubiquitin in DC.
4. the ubiquitinative FMDV protein antigen can be degraded by proteasome and can be processed in lysase. The antigenic peptide produced is presented to CD4+T cells or CD8+T cells in the way of cross extraction..CD4+T cells can only identify the MHC-II molecules carrying the antigen epitopes, so the content of IFN- gamma caused by lysosome inhibitors is significant. The decrease clearly indicates that DC can present FMDV antigen by the lysosome -MHC-II molecular pathway, and the strong inhibitory effect of proteasome inhibitor on the secretion of IFN- gamma by CD4+T cells suggests that there may be a mechanism for the cross presentation of FMDV antigen in the molecular pathway of proteasome -MHC-II. And CD4+T cells are activated by antigenic peptides. The main differentiation is Th1 cell subclass and secrete high level of IFN- gamma. From the above results, it is not difficult to see that the DC loaded by FMDV is mainly used as an antigen peptide of "endocytic body and lysosome -MHC-I molecular pathway" to present an antigen peptide to CD8+T cells, thus starting the CTL response. The ability of CD8+T cells to produce IFN- gamma is consistent with DC migration fatigue. The ability of CD8+T cells to produce IFN- gamma is 48h after being activated. Significant decline also showed a suppressed fatigue response.
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392

【参考文献】