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棘球蚴自然抗原和卡介苗对Th1/Th2转录因子和细胞因子的影响
Effect of Natural Antigens of Echinococcus Spp. and BCG on the Expression of Th1/Th2 Transcriptional Factor and Cytokine
【作者】 李红卫；
【导师】 杨玉荣；
【作者基本信息】 宁夏医科大学 ， 病原生物学， 2011， 硕士
【摘要】 目的模拟自然环境下泡球蚴感染的过程,通过观察Th1/Th2关键转录因子(T-bet和GATA-3)和其标志性细胞因子在卡介苗和棘球蚴自然抗原免疫及免疫后再用泡球蚴原头蚴攻击感染昆明鼠模型的表达水平,探讨包虫病感染对中间宿主的抗感染免疫和免疫病理反应在不同情况下的调节机制,并评估其对宿主细胞免疫反应的影响。方法建立昆明小鼠动物模型:用实验室常规制备的含0.1mg/ml蛋白的单房棘球蚴囊液抗原和泡球蚴组织提取抗原以皮下注射的方法免疫昆明小鼠,初始免疫量为0.2mg/每只,每一个月加强免疫量为0.1mg/每只。用市售的卡介苗(BCG)免疫方法同前(0.05mg/每只注射)。取一半的免疫后小鼠作泡球蚴原头蚴的攻击感染,感染方采用腹腔内注射2000个原头蚴(PBS稀释)。上述所有组各为20只鼠,雌雄各半。设PBS注射的空白对照和不作免疫的感染对照。各组10只雌雄各半。动物模型的处理和标本的收集:攻击感染四个月后,所有的鼠用乙醚麻醉处死前取血,然后立即取肝脾脏放入RNALater保存液中。处死后记录一般状况并检查个器官状态和感染病灶的分布,大小等情况;所取得的标本放置于-800C备用。用实时定量聚合酶链反应(qRT-PCR)检测脾组织中GATA-3及T-bet的mRNA表达水平;酶联免疫吸附法检测血清中白介素4(IL-4)、γ干扰素(IFN-γ)、白介素2(IL-2)、白介素6(IL-6)、粒细胞-巨噬细胞集落刺激因子(GM-CSF)、白介素10(IL-10)和白介素17(IL-17)的含量。所有的数据结果用SPSS软件做单因素相关分析,并对比个各组间转录因子和细胞因子的表达量均数差别的显著性检验。结果:泡球蚴攻击感染的小鼠在感染20天后,腹外可看到小米粒大的包囊,之后,腹腔逐步膨隆,活动减弱,毛发稀疏。同时出现互相撕咬现象,个别小鼠咬头,有些小鼠出现哮喘等过敏样症状,多数小鼠尿道口红肿,导致尿道水肿,排尿不畅,解剖发现膀胱充盈,肾脏灰白色,未做任何处理,很快死亡。所有实验鼠包括正常对照,感染对照,免疫和免疫后感染鼠模型的成活率为73% (102/140)并且各感染组病灶的大小和重量比较的差异无显著意义;Th1/Th2关键转录因子和细胞因子在血清中的表达量检测结果显示:比较CE免疫组、AE免疫组、卡介苗免疫组、感染对照组、CE免疫攻击组和AE免疫攻击组与PBS对照组小鼠的转录因子(GATA-3mRNA和T-betmRNA)和其标志性细胞因子(INF-γ及IL-4)以及其它细胞因子包括GM-CSF、IL-2、IL-6、IL-10和IL-17的表达量,差异均无统计学意义(P>0.05)。比较卡介苗免疫攻击组与PBS对照组的转录因子(T-betmRNA)和其标志性细胞因子(INF-γ)以及其它Th1/Th17型的细胞因子(GM-CSF、IL-2和IL-17)的表达量,差异有统计学意义(P<0.05)。而比较卡介苗免疫攻击组与PBS对照组及感染对照组的转录因子(GATA-3mRNA)和其标志性的细胞因子(IL-4)和其它Th2细胞因子(IL-6和IL-10)的表达量,差异无统计学意义(P>0.05)。卡介苗免疫攻击组T-betmRNA的表达量与IFN-γ成正相关(r=0.71,P<0.05)。结论: Th1/Th2细胞免疫反应在泡球蚴感染的昆明鼠动物模型宿主体内可以长期共同存在处于平衡状态,反映了宿主对泡球蚴产生的抗感染免疫和由寄生虫抗原诱导宿主产生的免疫病理反应的相互作用和相互适应的复杂调节过程。实验证明卡介苗(BCG)有上调Th1型免疫反应的作用,用BCG可以干预或治疗由泡球蚴抗原诱导的晚期泡球蚴(AE)病人的免疫抑制状态。
【Abstract】 Objective: To investigate the cellular immune effects of either immunization by natural antigens of Echinococcus spp. and BCG or challenge by the protoscolexes of E. multilocularis (Em) in mice model of Kunming spp.Methods: After either immunized mice with BCG (0.1 IU/ml), natural antigens (0.1mg/ml) of Echinococcus spp. By subcutaneous injection 0.2ml/per mice firstly, and boosted 0.1ml/per mice after a month for twice. After immunization above, half numbers of mice were challenged by use of E. multilocularis protoscolexes. Every immunized and challenged group had 20 mice with half and half females and males. The normal control and infectious control (no-immunization but only infectious) group had 10 mouse, respectively with half and half females and males. A quantity of the signature Th1/Th2 cytokines of IFN-γ/IL-4 and other cytokines (GM-CSF or IL-2/IL-6 and IL-10/ IL-17) in serum by using capture-ELISA, and of the transcriptional regulators of T-bet and GATA-3 in spleen-tissue by using qRT-PCR was undertaken. The data analysis were carried out by use of SPSS for a comparison of the average expression amounts of various proteins between different groups and the 95% CIs was set-up for significant difference.Results: After 20 days challenging of alveolar echinococcosis protoscolexes, the successful infectious mice (73%, 102/140) can be observed some allergic symptoms and abdominal lesion growth signs. Comparing the weight and size of lesions in various infectious group showed all in the insignificant findings. During the whole time infection-period after E.multilocularis protoscolex challenge, both key transcription factors and signature cytokines showed a homogeneious expression patterns. But it was very different in BCG immunized mice of either post-challenge or non-challenge, showing regulated the expressions of T-bet mRNA and signature cytokine of IFN-γand other Th1/Th17type of cytokines (IL-2, GM-CSF,IL-17) though insignificant impact of GATA-3 mRNA and its signature cytokine of IL-4 and or other cytokines ( IL-6/ IL-10) expressions were found.Conclusion: The long term balanced co-existence of Th1/Th2 reactions in mice model with alveolar echinococcosis (AE) infection may reflect a regulation mechanism for optimally adapted immune responses to a plethora of invading pathogens by adjusting a fine balance between protective and immunopathological inflammatory processes. BCG may be used for intervention when immune-suppression is induced in AE sufferance.