【作者】 王耀丽；

【导师】 钱桂生；

【作者基本信息】 第三军医大学 ， 内科学， 2007， 博士

【摘要】 目前,支气管哮喘（简称哮喘）已成为全球范围内变应性疾病中最常见的代表性疾病,发病率仍在持续增高,其发病机制尚未完全清楚,至今仍缺少有效而可行的措施来防治。20世纪80年代提出的“卫生学说”并不足以完全解释哮喘气道炎症的发生机制。但“卫生学说”强调了环境卫生因素对机体免疫反应的影响。近年的研究也发现,哮喘是一种对过敏原免疫耐受缺陷的疾患,其中树突状细胞（DC）可能是关键的调节因素之一。为此,本研究针对哮喘免疫耐受缺陷的复杂性和多样性,以及诱导免疫耐受途径的多样性。利用过敏原DNA疫苗诱导免疫耐受与Fc基因靶向DC诱导免疫耐受的两种策略结合起来,设计制备了具有高效免疫应答的重组变应原DNA疫苗(OVA-Fc-pcDNA_3.1)。探讨其在小鼠体内诱导特异性免疫耐受的效力和在小鼠遭遇变应原致敏模型中的免疫保护力,并研究OVA-Fc-pcDNA_3.1靶向DC在诱导致敏/激发小鼠对过敏原特异性免疫耐受的作用机制,将它们与OVA-pcDNA_3.1相比较,观察诱导免疫耐受的作用的异同,以及其在应用中的免疫安全性的问题。为深入开发新型重组变应原DNA疫苗奠定理论基础。研究内容和方法:1.构建OVA- Fc-pcDNA_3.1重组质粒,将pcDNA_3.1、OVA-pcDNA_3.1及OVA- Fc-pcDNA_3.1重组质粒大量抽提并进行去内毒素、去RNA纯化。在此基础上,重组质粒在CHO细胞中的体外转染与表达;鉴定OVA- Fc融合蛋白能否透过CHO细胞膜进入到培养上清液中,RT-PCR方法检测其瞬时表达,ELISA检测其在CHO细胞中的表达。MTT法检测重组质粒的细胞毒性。2. 4～6周健康BALB/c小鼠,雌雄不限,体重16-20g。将小鼠随机分为五组,每组6只,分次分批滚动喂养。1组为正常对照组;2组为哮喘组,以0.01M PBS（pH 7.2）代替质粒治疗小鼠;3组为去内毒素的OVA- Fc - pcDNA_3.1质粒治疗组;4组为去内毒素的OVA- pcDNA_3.1质粒治疗组,5组为去内毒素的pcDNA_3.1质粒治疗组,给予空白质粒治疗。末次激发后取肺脏组织行病理学检查,观察变应性气道炎症的H.E.变化。ELISA检测末次激发后小鼠血清中OVA特异性IgE抗体水平、BALF中细胞因子IL-2、IL-4、IL-5、IL-10、IFN-γ水平。3.观察OVA-Fc-pcDNA_3.1疫苗对哮喘小鼠外周血T细胞压群的影响。4～6周健康BALB/c小鼠,雌雄不限,体重16-20g。将小鼠随机分为五组,每组6只,分批滚动喂养。1组为正常对照组;2组为哮喘预防组,以0.01M PBS（pH 7.2）代替质粒预防哮喘小鼠组;3组为去内毒素的OVA-Fc-pcDNA_3.1质粒预防组;4组为去内毒素的OVA- pcDNA_3.1质粒预防组,5组为去内毒素的pcDNA_3.1质粒预防组,给予空白质粒预防。末次激发后流式细胞计量仪检测末次激发后小鼠外周血CD₄⁺、CD₈⁺T细胞分类情况。4.观察OVA-Fc-pcDNA_3.1疫苗对哮喘小鼠脾脏T淋巴细胞的影响。4～6周健康BALB/c小鼠,雌雄不限,体重16-20g。将小鼠随机分为五组,每组6只,分批滚动喂养。1组为正常对照组;2组为哮喘组,以0.01M PBS（pH 7.2）代替质粒治疗小鼠;3组为去内毒素的OVA-Fc-pcDNA_3.1质粒治疗组;4组为去内毒素的OVA- pcDNA_3.1质粒治疗组,5组为去内毒素的pcDNA_3.1质粒治疗组,给予空白质粒治疗。末次激发后流式细胞计量仪检测末次激发后小鼠脾脏T淋巴细胞中Foxp3表达水平。5.观察OVA-Fc-pcDNA_3.1疫苗对哮喘小鼠树突状细胞的影响。4～6周健康BALB/c小鼠,雌雄不限,体重16-20g。将小鼠随机分为五组,每组6只,分批滚动喂养。1组为正常对照组;2组为哮喘组,以0.01M PBS（pH 7.2）代替质粒治疗小鼠;3组为去内毒素的OVA-Fc-pcDNA_3.1质粒治疗组;4组为去内毒素的OVA- pcDNA_3.1质粒治疗组,5组为去内毒素的pcDNA_3.1质粒治疗组,给予空白质粒治疗。末次激发后流式细胞计量仪检测末次激发后小鼠脾脏、肺脏DC共刺激分子的表达水平。6.观察OVA-Fc-pcDNA_3.1疫苗对哮喘小鼠免疫安全性的研究。4～6周健康BALB/c小鼠。动物和分组4～6周健康BALB/c小鼠,雌雄不限,体重16-20g。将小鼠随机分为五组,每组6只,分批滚动喂养。1组为正常对照组;2组为哮喘组,以0.01M PBS（pH 7.2）代替质粒治疗小鼠;3组为OVA-Fc-pcDNA_3.1质粒治疗组;4组为OVA- pcDNA_3.1质粒治疗组,5组为pcDNA_3.1质粒治疗组,给予空白质粒治疗。6组为去内毒素的OVA-Fc-pcDNA_3.1质粒治疗组;7组为去内毒素的OVA- pcDNA_3.1质粒治疗组,8组为去内毒素的pcDNA_3.1质粒治疗组,给予去内毒素的空白质粒治疗。末次激发后,从病理组织学和免疫学角度观察重组质粒的免疫安全性问题。结果:1.所构建的OVA-Fc-pcDNA_3.1重组质粒分别经双酶切和PCR鉴定,以及DNA双向测序证实目的基因插入成功。采用RT-PCR方法能够从重组质粒转染的CHO细胞中扩增出与目的基因大小一致的DNA片断。ELISA分析表明在CHO细胞培养上清中可获得目的基因表达产物。2. OVA致敏/激发小鼠肺部呈现显著变应性炎症,BALF中细胞总数和嗜酸性粒细胞（EOS）比例显著增高,BALF和血浆中IL-4、IL-5水平显著升高,IFN-γ水平显著降低。3.将不同质粒皮下免疫哮喘小鼠后,能显著地减轻致敏/激发小鼠肺部变应性炎症,减少BALF中细胞总数和EOS比例、降低BALF中IL-4、IL-5的水平和增高IFN-γ水平,其中OVA -Fc - pcDNA_3.1免疫处理最为显著,与正常组无显著差异。4.将不同质粒皮下免疫哮喘小鼠后,流式细胞仪检测各试验组小鼠脾脏T淋巴细胞中Foxp3的表达有不同程度升高,其中OVA -Fc - pcDNA_3.1免疫处理后升高最为显著。5.将不同质粒皮下免疫哮喘小鼠后,小鼠外周血中CD₄⁺、CD₈⁺T细胞数有显著升高,其中OVA -Fc - pcDNA_3.1免疫处理最为显著。6.将不同质粒皮下免疫哮喘小鼠后,流式细胞仪检测各试验组小鼠肺脏树突细胞表面协同刺激分子CD11cCD80和CD11cCD86的表达有不同程度减低,其中OVA -Fc - pcDNA_3.1免疫处理后降低最为显著。7.没有用去内毒素纯化的质粒免疫小鼠后存在一定免疫性损伤的危险性,如过敏反应、急性肺损伤效应。去内毒素纯化的质粒经皮下免疫哮喘小鼠后,能在小鼠体内激发显著的特异性体液免疫和细胞免疫应答,有效地保护免疫后小鼠减轻哮喘的气道炎症,保证了免疫的有效性和安全性。结论:1.本研究利用基因工程技术,构建重组质粒OVA-Fc-pcDNA_3.1。经双酶切、PCR和DNA双向测序鉴定后,证实重组质粒构建成功。采用RT-PCR方法能够从重组质粒转染的CHO细胞中扩增出与目的基因大小一致的DNA片断。ELISA分析表明在CHO细胞培养上清中可获得目的基因表达产物。并利用去内毒素质粒大量抽提纯化试剂盒,使OVA-Fc-pcDNA_3.1疫苗系统更安全无毒性。2.将不同质粒皮下免疫哮喘小鼠后,均可在小鼠体内诱导特异性体液免疫、细胞免疫和粘膜免疫应答。其免疫应答和免疫保护作用均以去内毒素的OVA-Fc-pcDNA_3.1疫苗最强,且对异种感染攻击具有相同交叉免疫保护作用。3.OVA-Fc-pcDNA_3.1可能通过下调小鼠肺脏树突细胞表面协同刺激分子CD11cCD80和CD11cCD86的表达,并进一步诱导OVA致敏/激发小鼠的调节性T细胞的产生,从而诱导过敏原特异性的“免疫耐受”。4.去内毒素的OVA-Fc-pcDNA_3.1疫苗免疫哮喘小鼠后均未引起过敏反应、免疫损伤以及急性肺损伤效应,不存在诱发免疫损伤的潜在危险性。更多还原

【Abstract】 Background:Asthma is a chronic inflammatory disorder of the airways, characterized by Th2-dominated immune responses. Allergen gene vaccines have been shown to be effective in modulationing the allergic immune response in mouse models,especially shifting the immune response fron a Th2- to a Th1-dominated immune response. To enhance the effectivemess of DNA vaccination and potentially treat patients with ongoing airway inflammation,we constructed a DNA vaccination plasmid containing cDNA for a prototypic allergen,OVA,fused to the cDNA of a potent immune modulating Fc.This approach is based on fact that Fc,enhancing DCs’absorption,and is very powerful in driving the production of Th1 cytokine synthesis in na?ve and memory T cells.Objective:We investigated the combined effect of OVA and Fc vaccination on asthma development in BALB/c mice.It would lay a foundation for developing a new DNA vaccine.The aim of the study was to analyze whether dendritic cells targeted with allergen-DNA conjugates are able to stimulate autologous CD₄⁺ T cells, CD₈⁺ T cells,or both from atopic individuals to produce TH1 cytokines instead of TH2 cytokines.Methods:1. Animals BALB/c mice,6–8 weeks old at the onset of experiments and sex matched within each experiment.Mice care and use were performed in accordance with the guidelines of Dutch Committee of Animal Experiments.2.DNA constructs and purification The PCR products of OVA and Fc were selected as target DNA fragments and cloned into pcDNA_3.1 （+） to construct the recombinant plasmids OVA-pcDNA_3.1and OVA-Fc- pcDNA_3.1 respectively. The recombinant plasmids were constructed and expressed successfully in CHO cell lines. All Plasmids were purified by the EndoFree Plasmid Giga Kit.The recombinant plasmids were identified by restriction enzyme analysis and PCR reaction and confirmed by DNA sequencing.3.Study groups BALB/c mice were divided into five groups. 4.Immunization protocols Except for the control group,BALAB/c mice from all groups were first sensitized with OVA （gradeⅡ）before vaccination with the DNA plasmids.OVA （50μɡ） absorbed to alum was adminiatered i.p. once on day 0 . On days 8 and 9 the mice were challenged with aerosols of 1% OVA （gradeⅡ） diluted in PBS for 30 min each day.On days 10 and 25 the different endoFree DNA constructs were injected i.m. in the quadriceps muscles （100μɡin 100μl 0.9%Nacl）.On day 39 and 40 the mice were challenged with aerosols of 1% OVA （gradeⅤ） diluted in PBS for 30 min each day. Mice from control group received a mock sensitization with intraperitonealn alum alone and a mock challenge with aerosol PBS at the corresponding time. Mice were sacrificed within 1 days of the last OVA challenge. 5. Collection of bronchoalveolar lavage fluid（BALF） Mice were anesthetized with urethane 24 h after the last OVA challenge, and the abdominal cavity was opened. Blood samples for serum were collected from the vena cava . The tracheas were cannulated ,and BAL was performed by two lavages with 0.5 ml ice-cold PBS. The total cell number in BALF was determined. The BALF was centrifuged and supernatant used to test for cytokine prouduction and the cell pellet used to prepare slides for differential cell counting.Cytospin slides were fixed and stained with Diffquik for leucocyte differential analysis.and the number of monocytes, lymphocytes,and eosinophils in a total of 200 cells were counted in each slide. 6. Concentrations of cytokines in BALF were measured with commercial ELISA kits according to the manufacture’s instructions.Murine IFN-γ,IL-5 and IL-2,IL-10 ELISA kits.7.OVA-specific IgE assay Mice were bled et the time of sacrifice, and OVA-specific IgE was determined using a modified Ag-specific ELISA. 8. Generation of DCs from pulmonary and culture Pulmonary-derived DCs were enriched according to the published methods. The DCs were collected and immediately used in our assays. 9.Immunofluoresence and flow cytometry analysis Pulmonary-derived DCs were incubated with FITC-labeled CD_11c, PE -labeled anti-CD₈₀ （B7-1） MAb, PE-labeled anti-CD86 MAb on ice for 30 min and washed with PBS. Ten thousand cells were collected for each sample, and the data were analyzed with FACScan flow cytometer and CELLQUEST software. DCs of pulmonary express levels of the costimulatory molecules CD_11cCD₈₀ and CD_11cCD₈₆ surface marker.10.Flow cytometric analysis Autologous CD₄⁺ and CD₈⁺ T cells were stimulated with these targeted DCs,and proliferation and cytokine production were measured.Flow-cytometry assay was carried to detect the numbers of CD₄⁺ and CD₈⁺T cells in peripheral blood of mice at the 24 h after the last OVA challenge. FITC-labeled CD₄⁺ and CD₈⁺ Mab,hemolytic agentand IgG2a.11.Histopathology Formalin-fixed lungs were embedded in paraffin,sectioned in 6-μm thick slices,and stained with hematoxylin and eosin for routine hidtology. 12.Statistical analysis Results were shown as mean±SD.Statistical analysis was performed with the statistical software package SigmaStat.All assays were compared using ANOVA followed by least squares difference-t analysis.Results and methods:1.Histologic changes The inflammatory characteristics caused by OVA sensitization/challenge are shown in Figure 2-1, Figure 2-2, Figure 2-3, Figure 2-4, Figure 2-5. Histologic scores of peribronchiolitis,perivasculitis, alveolitis,and peribronchial eosinophilia in all groups with OVA sensitization/challeng (OVA, pcDNA_3.1,OVA-pcDNA_3.1,and OVA-Fc-pcDNA_3.1 groups) were significantly higher than that in the control group. Comparison among all groups with OVA sensitization/challenge showed that the OVA-Fc-pcDNA_3.1 group had significantly milder peribronchiolitis,perivasculitis,alveolitis, and peribronchial eosinophilia than other groups. 2.Cellular composition of the lung inflammatory responses In comparison with epithelial cells , which constituted the majorison of BAL cells in the control group,lymphocytes were the major inflammatory cells found in BALF from other groups that underwent OVA sensitization and challenge. The number of eosinophilsin the BALF of OVA, asthma, OVA-pcDNA_3.1,and OVA-Fc-pcDNA_3.1 groups were all significantly greater than in control group （p<0.01）. The number of total cells and eosinophils in the OVA-Fc-pcDNA_3.1 group was significantly lower than in the OVA, asthma,and OVA-pcDNA_3.1 groups, with the lymphocyte number significantly lower than in the asthma, pcDNA_3.1,and OVA-pcDNA_3.1 groups. However, there was no difference in the number of monocytes and neutrophils between the four groups 3.Serum OVA-specific IgE OVA-sensitized/challenged mice (asthma, pcDNA_3.1 , OVA-pcDNA_3.1,and OVA-Fc-pcDNA_3.1 groups) showed significantly higher serum OVA-specific IgE titers than control mice. There were significant differences among all of the OVA-sensitized or–challenged groups.OVA-specific IgE was very high in OVA-immunized BALB/c mice.Vaccination with the different DNA vectors after immunization with OVA significantly reduced the level of OVA-specific IgE. The inhibitory effect on IgE production was strongest with the OVA-Fc fusion construct..4.Cytokine production in BALF The levels of IFN-γ,IL-2,IL-5,and IL-10 in BALF were assayed by ELISA.As showed in Figure 5, all OVA-sensitized/challenged groups (asthma, pcDNA_3.1, and pcDNA_3.1/OVA groups) had significantly lower IL-10,IL-2,IFN-γand higher IL-5 level than the control.IL-10 in OVA-Fc- pcDNA_3.1 group was significantly higher than in the asthma, pcDNA_3.1,and OVA-pcDNA_3.1 groups. 5. The numbers of CD₄⁺ and CD₈⁺ T cells in OVA-Fc- pcDNA_3.1 groups were higher significantly than in the asthma, pcDNA_3.1and OVA-pcDNA_3.1 groups.6.CD11cCD80 and CD11cCD86 were up-regulated on pulmonary-derived DCs from BALB/c mice sensitized and challenged with OVA. Vaccination with OVA-Fc- pcDNA_3.1 resulted in a dramatic decrease of CD11cCD80 and CD11cCD86. targeted DCs of pulmonary express lower levels of the costimulatory molecules CD11cCD80 and CD11cCD86 compared with that of asthma DCs.Conclusion:1.DNA sequencing and restriction endonuclease digestion analysis indicated that the eukarytotic expression vector OVA-Fc-pcDNA_3.1 had been concess of this plasmid constructed successfully.OVA-Fc expression could be detected in CHO cells by Western blotting,ELISA,and flow cytometry.2.EndoFree OVA-Fc-pcDNA_3.1 decreased asthmatic inflammation in OVA-ensitized/challended mouse model .Moreover,our studies,demonstrating that endoFree OVA-Fc fusion constructs have much greater immunogenicity than allergen-only cDNA constructs,suggest that EndoFree allergen-Fc DNA constructs may provide,effective,and potentially curative therapy for allergic disease and asthma.DCs can be targeted with OVA-DNA conjugates very efficiently by using Fc vector yielding DCs with high T-cell stimulatory capacities,directing the atopic-allergic immune response from Th2 dominance toward Th1 dominance.3.The data demonstrated that EndoFree OVA-Fc- pcDNA_3.1 recombinant plasmid inhibits lung inflammation and serum IgE in vivo. This effect may be due to reduced expression of CD11cCD80 and CD11cCD86 by DCs.4.EndoFree OVA-Fc DNA vaccine not only can enhance immunity effect of vaccine but also is safe and innocuous to mice.更多还原