包虫病所致过敏反应中免疫耐受机制的实验研究

发布时间：2018-06-21 08:44

  本文选题：免疫耐受 + 包虫病　； 参考：《新疆医科大学》2016年博士论文

【摘要】：目的:包虫病是全球人畜共患性疾病。包虫病所致的过敏性休克是其严重并发症,占包虫病患者突然死亡原因的20%。包虫病患者存在免疫耐受已被证实,但是这种状态在包虫囊液外溢时发生转变,过敏性休克发生,具体机制尚待阐明。为此,本研究在完善囊型包虫病所致过敏反应动物模型、优化小鼠过敏反应评价体系的基础上,探讨以Treg细胞为核心的“免疫耐受调节网络”中相关重要因子在囊型包虫病所致过敏反应中的变化及特点,以及地塞米松预处理对囊型包虫病所致过敏反应的影响及机制,为制定针对囊型包虫病所致过敏反应的有效防治策略奠定理论基础。方法:1)羊源细粒棘球蚴原头节及囊液均采自包虫感染羊肝脏。包虫感染组,每只小鼠通过腹腔接种粒棘球蚴原头节2000个,建立囊型包虫感染模型,再应用羊源细粒棘球蚴粗制囊液0.1ml/10g攻击发敏建立囊型包虫病所致过敏反应模型。通过观察过敏症状、肛温变化、肺组织HE染色病理切片,测定肺泡灌洗液中白细胞分类计数、过敏介质组胺及PAF-AH水平,测量血清抗体水平等一系列指标,全面优化过敏评价体系,完善过敏模型建立;2)BALB/c小鼠随机分成健康对照组、包虫感染组、囊液致敏组。使用全面的过敏评价体系评价过敏模型。FCM检测小鼠淋巴结来源PBMCs中DCs、Treg细胞水平,ELISA检测血清IgE、IgG、IgG1、IL-10、TGF-β1、IL-13、IL-17A水平;3)C57BL/6小鼠随机分成健康对照组、单纯囊液致敏组,地塞米松预处理组。在包虫囊液致敏前30min腹腔注射地塞米松建立地塞米松预处理模型。使用全面的过敏评价体系评价过敏模型。FCM检测小鼠淋巴结来源PBMCs中Treg细胞水平,ELISA检测血清IgE、IgG、IgG1、IL-10、TGF-β1、IL-13水平。结果:1)BALB/c小鼠包虫接种成功率为75%,C57BL/6小鼠包虫接种成功率为65%,囊型包虫病所致过敏模型成功率为分别为93.33%和85.62%。发生过敏反应小鼠出现了不同程度的过敏症状、肛温下降、肺组织HE病理切片及BALF中炎性细胞增加、组胺表达量增加、血清抗体IgE、IgG、IgG1水平增加。上述指标均表明BALB/c和C57BL/6小鼠囊型包虫病所致过敏反应模型建立成功;2)与健康对照组相比,包虫感染组及囊液致敏组小鼠血清IgE水平均明显增加(P0.01)。囊液致敏组小鼠血清IgE水平明显高于包虫感染组(P0.05)。囊液致敏后,肺组织病理及BALF中炎性细胞增加、组胺表达水平增加、血清IL-13、IL-17A水平增加(P0.05)。与健康对照组相比,小鼠血清IL-10、TGF-β1水平及CD4+CD25+Foxp3+Treg/CD4+细胞比例在包虫感染组增加,在囊液致敏组下降(P0.001),呈现先升后降的双向变化。但是其他T细胞群,如CD4+CD25+Foxp3-细胞比例、CD4+Foxp3+CD25-细胞比例,各组间比较无明显差异。CD11c+IA/IE+CD86+细胞比例、CD11c+IA/IE+CD80+细胞比例以及CD4/CD8比例,各组间比较无明显差异;3)小鼠严重过敏反应发生率在地塞米松预处理组是12.5%,单纯囊液致敏组是37.5%。与单纯囊液致敏组相比,地塞米松预处理组小鼠肺组织及BALF中炎性细胞降低、组胺表达水平减少、IgE水平降低、血清IL-13水平降低(P0.001)。血清IL-10及TGF-β1水平与CD4+CD25+Foxp3+Treg/CD4+细胞比例变化趋势相同,均在单纯囊液致敏组下降,在地塞米松预处理组增加。结论:1)全面的小鼠过敏评价体系优化了囊型包虫病所致过敏反应小鼠动物模型,为探讨包虫病所致过敏反应中免疫耐受机制奠定基础;2)以Treg细胞为核心的“免疫耐受调节网络”维持了包虫感染后的免疫耐受状态,细粒棘球蚴囊液致敏后,网络失衡,诱发过敏反应;3)包虫感染后以Treg细胞为核心的“免疫耐受调节网络”通过CD25、Foxp3高表达,IL-10、TGF-β1高分泌水平,维持免疫耐受功能。囊液致敏后,免疫耐受状态发生转变,Treg细胞比例下降,IL-10、TGF-β1水平降低,IL-17A、IL-13水平升高,发生过敏反应;4)在包虫病所致的过敏反应中,地塞米松可通过降低IgE水平,减少肺组织及BALF中炎性细胞渗出,减少靶器官过敏介质组胺释放,减轻过敏反应;5)地塞米松通过上调Treg细胞比例及功能维持“免疫耐受调节网络”功能。其具体机制是上调IL-10和TGF-β1水平,抑制Th2细胞因子IL-13水平。
[Abstract]:Objective: hydatid disease is a global zoonotic disease. Hydatid disease caused by anaphylactic shock is the serious complications, accounted for 20%. of hydatid disease hydatid disease suddenly causes of death in patients with the presence of immune tolerance has been confirmed, but this change in hydatid fluid overflow, allergic shock, tillneeds to clarify. In this study, due to cystic echinococcosis anaphylaxis animal model improvement, optimization based on the evaluation system of anaphylaxis in mice, to investigate the Treg cells as the core of the "immune tolerance regulation network related important factor in cystic hydatid disease caused by allergic reactions in changes and characteristics, and the effect of dexamethasone pretreatment on cystic echinococcosis disease The impact caused by the allergic reaction and mechanism, which lays the theoretical foundation for the formulation of effective prevention strategies for cystic hydatid disease caused by an allergic reaction. Methods: 1) sheep derived Echinococcus granulosus protoscolex and cyst fluid were collected from sheep liver hydatid infection. Infection group, each mouse by intraperitoneal inoculation of grain granulosus protoscolex 2000 and the establishment of cystic echinococcosis Infection model using sheep derived Echinococcus granulosus cystic fluid 0.1ml/10g attack caused by a sensitive cystic echinococcosis allergic reaction model. Through the observation of allergic symptoms, changes of rectal temperature, HE staining of lung tissue pathological sections in bronchoalveolar lavage fluid in white blood cell count, allergic medium histamine and PAF-AH levels, the measurement of serum antibody level etc. A series of indexes, comprehensive optimization of allergy evaluation system, improve the allergy model; 2) BALB/c mice were randomly divided into control group, infection group, allergy group. The cystic fluid evaluation system of comprehensive evaluation of the use of allergy allergy model.FCM mice were detected from lymph nodes in PBMCs DCs and Treg cell levels, serum IgE, ELISA, IgG IgG1, IL-10, TGF-, beta 1, IL-13, IL-17A; 3) C57BL/6 mice were randomly divided into healthy control group, simple cystic fluid sensitized group, dexamethasone pretreatment group. In hydatid fluid before sensitization by intraperitoneal injection of 30min dexamethasone to establish dexamethasone pretreatment model. Using the evaluation system of comprehensive evaluation of allergic allergy detection model.FCM mouse lymph node cell Treg PBMCs in The level of serum IgE, ELISA, IgG, IgG1, IL-10, TGF- beta 1, IL-13 1). Results: the success rate of BALB/c mice inoculated with echinococcosis was 75%, the success rate of C57BL/6 mice inoculated with 65% hydatid cystic hydatid disease caused by allergy, the success rate of the model was respectively 93.33% and 85.62%. mice developed allergic allergic reaction the symptoms of different degree, rectal temperature decreased, The inflammatory cells of HE pathology and BALF in lung tissue increased, the increase in expression of histamine, serum antibody IgE, IgG, IgG1. The above indexes showed increased levels of BALB/c and C57BL/6 in mice caused by cystic echinococcosis allergic reaction model; 2) compared with healthy control group, infection group and hydatid cyst fluid sensitized group serum IgE levels were significantly increased Plus (P0.01). The cystic fluid sensitized mice serum IgE levels were significantly higher than that of hydatid infection group (P0.05). Sensitized cystic fluid, lung tissue pathology and BALF in inflammatory cells increased, the expression level of histamine increased, serum IL-13, IL-17A levels increased (P0.05). Compared with the healthy control group, serum IL-10 rat, TGF- beta 1 and CD4+CD25+Foxp3+Treg/CD4+ cell ratio In cases of hydatid infection group, sensitized group in cyst fluid decreased (P0.001), shows the first increased and then decreased. But the other T cells, such as CD4+CD25+Foxp3- cell ratio, CD4+Foxp3+CD25- cell ratio,.CD11c+IA/IE+CD86+ cell ratio is no significant difference between groups, the proportion of CD11c+IA/IE+CD80+ cells and the ratio of CD4/CD8, ratio between groups There are no significant difference; 3) severe allergic reactions in mice in dexamethasone pretreatment group is 12.5%, the Dan Chunnang solution sensitized group is 37.5%. with simple cystic fluid sensitized group, dexamethasone pretreatment of inflammatory cells in lung tissues of mice in BALF and decreased the expression level of histamine decreased, the decrease of IgE level, the level of serum IL-13 lower serum I (P0.001). L-10 and TGF- beta 1 level and the percentage of CD4+CD25+Foxp3+Treg/CD4+ cells in the same trend, both in pure cystic fluid sensitized group decreased, increased in the dexamethasone treated group. Conclusion: 1) a comprehensive evaluation system to optimize the mouse allergic reaction caused by cystic hydatid disease in mice animal model, to explore the immune induced allergic reaction in hydatid disease resistance By laying the foundation mechanism; 2) to Treg cells as the core of the "tolerance regulation network" to maintain the infection after immune tolerance, sensitization of Echinococcus granulosus cyst fluid, network imbalance, cause an allergic reaction; 3) hydatid after infection with Treg cells as the core of the immune tolerance regulation network by CD25. The high expression of Foxp3, IL-10, TGF- beta 1 High level, maintain tolerance function. The cysts after sensitization, immune tolerance state changes, the proportion of Treg cells decreased IL-10, TGF- beta 1 reduced the level of IL-17A, IL-13 increased the level of allergic reaction; 4) in hydatid disease caused by allergic reaction, dexamethasone can reduce IgE level, reduce inflammatory cell infiltration in lung tissue and BALF Out, reduce target organ allergy mediators histamine release, reduce allergic reactions; 5) dexamethasone through upregulation of Treg cell proportion and maintain the function of "immune tolerance regulation network function. Its mechanism is upregulated IL-10 and TGF- beta 1 level, the level of Th2 cell inhibitory factor IL-13.
【学位授予单位】：新疆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R532.32
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本文编号：2047935