细粒棘球绦虫抗原Fis1T细胞表位肽缓解过敏性哮喘小鼠气道炎症的免疫学作用研究

Immunological effect of T cell epitope peptide of Fis1 antigen from Echinococcus granulosus on alleviating airway inflammation in allergic asthma mice

目的探讨细粒棘球绦虫Fis1蛋白的T细胞表位肽(Eg.Fis183-102)对过敏性哮喘小鼠气道炎症的缓解作用及免疫学作用机制。方法1)体内试验:将24只6~8周雌性BALB/C小鼠随机分为3组(每组8只),其中B组为对照组,C组为过敏性哮喘组(OVA),T组为干预组(OVA+Eg.Fis183-102)。采用ELISA法检测小鼠血清OVA特异性IgE水平;采用HE,PAS,MASSON组织染色检查小鼠肺组织病理变化;采用流式细胞术分析肺脏和脾脏组织中嗜酸性粒细胞比例、Th1和Th2的分群及比例。2)体外试验:采用裂红法分离小鼠脾脏单个核细胞,每孔以1×106个细胞铺板,分为3组(每组3个复孔),其中B组为对照组,C组为刀豆蛋白刺激组(ConA),T组为干预组(ConA+Eg.Fis183-102),培养3 d后收集上清,采用ELISA法检测IFN-γ和IL-4水平。结果1)体内试验:与C组相比,T组小鼠肺组织的炎细胞浸润、胶原沉积等病理损伤有所改善,血清OVA特异性IgE水平、肺组织嗜酸性粒细胞比例、肺脏和脾脏Th2细胞比例均显著下降(均P<0.05),肺和脾Th1细胞比例显著上升(均P<0.05)。2)体外试验:与B组相比,C组小鼠脾脏单个核细胞培养上清中IFN-γ和IL-4水平均显著上升(均P<0.05);与C组相比,T组脾脏单个核细胞培养上清中IFN-γ水平显著上升,IL-4水平显著下降(均P<0.01),与体内试验结果一致。结论Eg.Fis183-102可缓解过敏性哮喘小鼠肺组织炎症损伤,降低IgE的分泌,并可能通过纠正Th1/Th2免疫应答失衡抑制OVA诱导的过敏性哮喘小鼠的气道炎症。

Objective Exploring the alleviative effect and immunological mechanism of T cell epitope peptide(Eg.Fis1_(83-102))of Fis1 protein from Echinococcusgranulosuson airway inflammation in allergic asthma mice.Methods 1)In vivo:24 female BALB/C mice at 6-8 weeks were randomly divided into 3 groups(8 mice per group),Group B is the control group,group C is the allergic asthma group(OVA)and group T is the intervention group(OVA+Eg.Fis1_(83-102)).Measurement of serum OVA-specific IgE levels in mice by ELISA;Detection of histopathological changes in mouse lung using HE,PAS,MASSON tissue staining;Flow cytometry was used to analyse the fractions and ratios of eosinophils,Th1 and Th2 in lung and spleen tissues.2)In vitro:Isolation of single nucleated cells from mouse spleen by the red cleavage method,Each well was plated with 110~6 cells and divided into 3 groups(3 replicate wells per group).Group B is the control group,group C is the knife-bean protein stimulation group(ConA)and group T is the intervention group(ConA+Eg.Fis1_(83-102)).The supernatant was collected after 3 d of incubation and the levels of IFN-γand IL-4 in the culture supernatant were measured by ELISA.Results 1)In vivo:Compared with group B,mice in group C had a large number of inflammatory cells infiltrating around the airway,collagen deposition and other pathological damage,Bronchial inflammation score(0.333±0.5774 vs 3.667±0.5774,P<0.01),PAS score(0.333±0.5774 vs 3.333±0.5774 P<0.01),and MASSON score(2.470±0.1975 vs 6.942±0.1393,P<0.0001).Pathological damage in lung tissue of mice in group T was ameliorated compared to group C,Bronchial inflammation score(3.667±0.5774 vs 1.333±0.5774,P<0.01),PAS score(3.333±0.5774 vs 1.667±0.5774,P<0.01),MASSON score(6.942±0.1393 vs 3.270±0.1220,P<0.01).Compared with group B,Serum OVA-specific IgE levels(0.4869±0.04563 vs 0.7447±0.1786),the proportion of eosinophils in lung tissues(1.580±1.038 vs 7.348±1.151),and the proportions of Th2 cells in lungs and spleens(0.3635±0.2602 vs1.243±0.3308,0.6150±0.1323 vs1.583±0.2103)were elevated of mice in group C(all P<0.01).Compared with group C,Serum OVA-specific IgE levels(0.7447±0.1786vs 0.4556±0.01743),the proportion of eosinophils in lung tissues(7.348±1.151vs 5.233±1.268),and the proportions of Th2 cells in lungs and spleens(1.243±0.3308vs 0.4033±0.1429,1.583±0.2103vs0.8150±0.1060)were decreased of mice in group T(all P<0.01).Compared with group C,the proportions of Th1cells in lungs and spleens(2.933±0.2801 vs3.930±0.5303,3.025±0.2931 vs 4.950±0.3803)were elevated of mice in group T(all P<0.05).2)In vitro:IFN-γlevels were significantly increased in the culture supernatants of splenic mononuclear cells in group T compared with mice in group C(81.69±13.39 vs 133.1±6.074,P<0.05),IL-4 levels decreased significantly(148.4±5.069 vs 119.7±5.859,P<0.01),consistent with the results of the in vivo test.Conclusion Eg.Fis1_(83-102)alleviates inflammatory damage in lung tissue of allergic asthmatic mice,decreases IgE secretion and suppresses OVA-induced airway inflammation in allergic asthmatic mice possibly by modulating Th1/Th2 immune response imbalance.