囊型包虫病致过敏性休克特异性抗原的识别、纯化与免疫原性研究

Hydatid disease has a high incidence in western China area, and its infection rate is about 2-12%. Anaphylactic shocks (AS) induced by hydatid disease is a serious complication during peri-operation, and the incidence rate is about 4.6%. Patients with echinococcosis-caused AS usually have poor responses and high mortality to treatments for type I hypersensitivities. Based on our previous study showed that: IgE, IgG and IgG1 levels in AS patients were significantly elevated compared to non-AS Hydatid disease patients; meanwhile, proteins with high molecular weight antigens in the Echinococcus granulosus cyst fluid (EgCF) can be recognized by anti-hydatid specific IgE. Based on these observations, we made a hypothesis that some specific protein antigens in EgCF are allergens which can induce AS. However, the protein components in the EgCF are very complicated, so the identification and purification of specific allergens which induce AS are critical for the research about AS immune responses and for the applications of clinical diagnosis and treatment. This grant is continuing of our previous studies, and it will use serum samples from AS Hydatid patients to explore the allergens which induce the AS, and to explore the allergen-induced humoral and cellular immune responses. The isolated allergens will be subjected to the development of reagent kit for quick and convenient screen of possible echinococcosis-caused AS patients. Moreover, the identified AS-induced allergens can be used for the generation of immunoglobulins for allergen neutralization. The anticipated results may provide essential insights into the development of new therapeutic strategies for prevention and management of CE (Echinococcosis cyst)-induced anaphylactic shocks.

包虫病由细粒棘球绦虫引起，西部高发，感染人群达2-12%。包虫病所致过敏性休克（anaphylactic shock; AS）为该病严重并发症，发生率达4.6%，采用抗Ⅰ型过敏反应药物进行治疗效果不佳，死亡率高。我们发现：AS患者IgE，IgG和IgG1水平明显升高，且囊液粗制抗原中较大分子量的免疫原性蛋白（43kDa）能与抗棘球蚴特异性IgE结合，可诱发包虫病感染动物AS。因此认为囊液中具有特定免疫原性的蛋白质是导致AS的抗原分子。但粗抗原成份复杂、含有多种蛋白组份，所以特异性抗原识别与纯化成为AS免疫机制研究和临床诊治的关键。本课题利用收集的AS患者血样标本，识别并纯化特异性抗原；分析患者血液中免疫细胞；观察该抗原诱导的AS抗体与细胞免疫反应特点。纯化后的抗原将运用于发展快速、简便的术前筛检试剂盒及量化生产可中和致AS的特异性抗原的免疫球蛋白，为包虫病致AS的防治奠定科学基础。

包虫病由细粒棘球绦虫引起，西部高发，感染人群达2-12%。包虫病所致过敏性休克该病严重并发症，发生率达4.6%，采用抗Ⅰ型过敏反应药物进行治疗效果不佳，死亡率高。本课题利用收集的AS患者血样标本，识别并纯化特异性抗原；分析患者血液中免疫细胞；观察该抗原诱导的AS抗体与细胞免疫反应特点。利用收集的 AS 患者血样标本，分析患者血液中免疫细胞；观察该抗原诱导的 AS 抗体与细胞免疫反应特点。结果发现：sIgE的主要EgCF抗原为分子量约40.5kDa等电点PH约6.5-9.0的4个蛋白点；sIgG1的EgCF抗原为分子量约35.5kDa等电点PH约6.5-9.0的5个蛋白点。棘球蚴病所致过敏性休克不同于Ⅰ型变态反应，除IgE水平升高外，还同时伴随有IgG1水平和细胞毒介质水平的升高。若全身多发细粒棘球蚴感染，且包虫类型为CE2的多子囊型，包虫成熟度较低（无钙化及坏死），手术前血常规提示嗜酸性粒细胞和嗜碱性粒细胞含量过高，包虫八项实验室检查中抗EgCF 抗体、抗EgB 抗体为强阳性（+++），术前血清IgG水平较高者，再行手术治疗前需要谨防过敏性休克的发生，且有二次发生过敏性休克的可能。在小鼠模型中发现以Treg细胞为核心的细胞群，通过高表达Foxp3、CD25，上调IL- 10、TGF-β1水平，抑制IL-17及TH2类细胞因子IL-13而发挥免疫耐受功能，防止过敏反应发生。包虫感染可促进这群具有抑制作用分子水平的上升，免疫耐受得以维持。羊源粗制细粒棘球蚴囊液促使FOXP3核转位，细胞群中各细胞水平下降，过敏反应发生。