【作者】 王颖；

【导师】 陈建军；

【作者基本信息】 华中科技大学 ， 耳鼻咽喉科， 2019， 硕士

【摘要】 第一部分建立体内不同剂量过敏原局部滴鼻小鼠过敏性鼻炎模型【目的】目前建立变应性鼻炎动物模型有多种方法,其中最经典的方法是通过腹腔注射(Intraperitoneal Injection,IP)致敏加上局部激发,而最贴近自然的方法是仅通过局部滴鼻的方法。然而对于自然途径的动物模型还没有完全研究清楚,并且两种模型之间存在的差异也没有被完全理解。同时针对自然途径的动物模型中过敏原剂量反应关系仍不清楚。【方法】本研究采用局部滴入OVA(无佐剂,每日1次,持续9周)致敏小鼠,建立局部滴鼻AR模型。使用了5种不同剂量的OVA来研究过敏严重程度与过敏原的剂量的相关性。记录过敏性症状、检测脾脏细胞培养上清的细胞因子(IL-4、IL-5、IFN-r)、血清抗体(IgE、Ig G2a、Ig G1)、鼻腔灌洗液(Nasal Lavage Fluid,NALF)细胞因子(IL-4、IL-5、IFN-r)的浓度、观察鼻黏膜嗜酸性粒细胞、肥大细胞及杯状细胞的浸润。【结果】与空白组相比,除了0.0025 mg/ml OVA剂量组的打喷嚏次数、血清抗体浓度、细胞因子浓度和浸润嗜酸性粒细胞均明显增高。不同剂量过敏原浓度与过敏的严重程度有关。在一定浓度范围内,过敏症状的严重程度、IgE抗体水平、体内的IL-4、IL-5的浓度与接触过敏原浓度呈正相关,即随着接触的OVA浓度越高,过敏反应更倾向Th2反应。同时,经典IP注射组血清抗体(IgE、Ig G1)和细胞因子(脾细胞上清IL-4、IL-5和NALF的IL-4)明显高于滴鼻组,即与局部滴鼻模型相比,经过腹腔注射加局部激发的过敏性鼻炎模型更倾向于Th2反应。【结论】两种经典AR模型中,IP注射模型和局部滴鼻模型可引起不同免疫反应。在25mg/ml到0.025 mg/ml之间,局部滴鼻模型呈剂量-反应正相关性。第二部分体外研究不同剂量过敏原对未成熟树突状细胞的作用机制【目的】通过自然过敏性鼻炎动物模型研究得出不同剂量变应原刺激小鼠产生不同程度的过敏反应,而在过敏反应中变应原只可能先通过DC的抗原呈递作用后才能触发后续的反应。因此DC的不同功能状态,对后续的na?ve T细胞分化方向可产生极大影响。研究报道不同剂量过敏原对T细胞的作用是通过改变呈递的抗原量来发挥作用,而对于抗原剂量如何影响树突状细胞的研究极少。因此我们推测过敏原剂量是先通过产生不同功能状态的DC并提呈给na?ve T细胞不同抗原量进而诱导其向不同方向分化。【方法】体外分离培养成熟树突状细胞,用不同剂量过敏原刺激24h后,流式检测细胞表面抗原的表达,用RNA-seq技术分析不同剂量刺激后差异因素。用不同剂量抗原刺激后的DC与分选的na?ve T细胞共培养72h,流式检测各T细胞Th1(CD4+IL-4+)、Th2(CD4+IFNr+)、Th17(CD4+IL-17+)、Treg(CD4+CD25+Foxp3+)的表达情况。【结果】本研究发现,树突状细胞成熟标志物CD40、CD11c、CD86、MHC-Ⅱ、CD80的阳性率随着OVA剂量增大而升高,并且用不同剂量变应原刺激DC后,RNA-seq显示有多种关键指标有明显差异。不同剂量变应原诱导的树突状细胞与na?ve T细胞共培养后,流式检测结果显示10ug组,CD4+IL-4+细胞比例较高;100和1000ug组,CD4+IFN-r+细胞比例较高;10000ug组,CD4+CD25+Foxp3+细胞比例增高,CD4+IL-4+和CD4+IFN-r+细胞比例均下降。【结论】不同剂量变应原可刺激产生不同成熟状态的树突状细胞,并决定初始T细胞分化方向,微弱的过敏原刺激的DC即可诱导na?ve T细胞向Th2方向分化,当DC接触的抗原剂量升高时即可诱导na?ve T细胞向Th1方向分化,当DC接触的抗原超过一定剂量时会诱导na?ve T细胞分化产生Treg细胞。更多还原

【Abstract】 Part I: Establish the model of allergic rhinitis in mice with different dose of allergenObjective: Predominantly,two animal models are used for allergic rhinitis(AR),which are established by intraperitoneal(IP)injection plus local challenge and nasal-only delivery.The differences between these two models are not fully understood.Moreover,dose-response relationship to allergens remains unclear.Methods: In this study,mice were sensitized by nasal drops(without adjuvant,once daily for 9 weeks)to set up a nasal-only delivery AR model.Five different doses of OVA nasal drops were served to explore the dose-response to allergens.Allergic symptoms,serum antibodies(IgE,Ig G2 a,Ig G1),spleen supernatant and NALF(Nasal Lavage Fluid)cytokines(IL-4,IL-5,IFN-r)and infiltrated eosinophils of the nasal mucosa were observed.Results: The allergic symptoms,serum antibodies,cytokines and infiltrated eosinophils were significantly higher in the high OVA concentration compared with those of the control group.Different OVA concentrations associated with the severity of allergy.Within a certain concentration range,OVA concentration positively related to the severity of symptoms,IgE antibody level and Th2 bias.Meanwhile,serum antibodies(IgE,Ig G1)and cytokines(IL-4,IL-5 in spleen and IL-4 in NALF)were significantly higher in the classical IP injection group than in the nasal drip groups.Conclusion: The IP injection model and the nasal-only delivery model are two typical models for AR that causes different immune response.A positive dose-response relationship in the nasal-only delivery model is observed from 25 mg/ml to 0.025 mg/ml.Part II: The mechanism of action of different doses of allergens on immature dendritic cells in vitroObjective:According to the animal model of natural allergic rhinitis,different doses of allergen stimulated mice to produce different degrees of allergic reactions.In allergic reactions,allergens may only be triggered by DC antigen presentation before triggering subsequent reaction.Therefore,the different functional states of DC can have a great impact on the subsequent differentiation direction of na?ve T cells.Studies have reported that the effects of different doses of allergens on T cells play a role by altering the amount of antigen presented,and little is known about how antigen dose affects dendritic cells.Therefore,we speculate that the allergen dose is first induced by different DCs of different functional states and presented to different levels of na?ve T cells to induce differentiation in different directions.Methods:Mature dendritic cells were isolated and cultured in vitro.After stimulation with different doses of allergen for 24 hours,the expression of cell surface antigen(CD40、CD11c、CD86、MHC-Ⅱ、CD80)was detected by flow cytometry.The difference factors of different doses of stimulation were analyzed by RNA-seq technique.DCs stimulated with different doses of antigen were co-cultured with sorted na?ve T cells for 72 h.Flow cytometry was used to detect Th1(CD4+IL-4+)、Th2(CD4+IFN-r+)、Th17(CD4+IL-17+)、Treg(CD4+CD25+Foxp3+).Results:This study found that the positive rates of dendritic cell maturation markers CD40,CD11 c,CD86,MHC-II,and CD80 increased with increasing OVA dose,and RNA was stimulated with different doses of allergens.RNA-seq shows a number of key metrics that differ significantly.After co-culture of dendritic cells induced by different doses of allergens with na?ve T cells,the flow detection results showed that the ratio of CD4+IL-4+ cells was higher in the 10μg group and the ratio of CD4+IFN-r+ cells in the 100μg and 1000μg groups.Higher;in the 10000μg group,the proportion of CD4+CD25+Foxp3+ cells increased,and the proportion of CD4+IL-4+ and CD4+IFN-r+ cells decreased.Conclusion:Different doses of allergens can stimulate dendritic cells with different mature states and determine the direction of initial T cell differentiation.The weak allergen-stimulated DC can induce na?ve T cells to differentiate into Th2 direction.When the antigen dose is increased,the na?ve T cells can be induced to differentiate into Th1.When the antigen exposed by the DC exceeds a certain dose,the na?ve T cells are induced to differentiate into Treg cells.更多还原