自然感染细粒棘球蚴对肠道粘膜免疫屏障的影响研究

发布时间：2018-01-27 17:27

本文关键词： 寄生虫免疫细粒棘球蚴免疫因子肠粘膜免疫屏障　出处：《新疆医科大学》2016年博士论文　论文类型：学位论文

【摘要】：背景:由细粒棘球绦虫的幼虫引起的棘球蚴病,是一种人畜共患的寄生虫病,高发于我国及世界各地的畜牧区,分布地域相当广阔,目前已成为全球性的公共卫生问题。人类及部分食草类动物(如绵羊等)因为误食了由其终末宿主排出的虫卵成为其中间宿主,虫卵经过胃,最终在十二指肠及小肠上段肠粘膜内寄居,在此虫卵孵化出六钩蚴,穿过肠粘膜屏障入血并到达肝脏及其他器官,对机体的健康危害极大。既往研究发现,细粒棘球蚴之所以能够在机体内长期生存,是其通过影响宿主Th1及Th2两型细胞免疫应答平衡因子,诱导非保护性的Th2型免疫应答成为主导,利于细粒棘球蚴在体内的生长。但是,肠粘膜免疫屏障系统是机体内最大的淋巴免疫系统,是细粒棘球蚴进入机体的第一道屏障,其对肠粘膜免疫屏障影响的相关研究鲜有报道,本研究旨在通过自然感染细粒棘球蚴的绵羊做为大动物研究对象,采取随机对照实验,应用酶联免疫吸附测定(ELISA)及免疫组化等方法,分析Th1/Th2相关的部分免疫因子(IL-6,IL-10及IFN-γ)在外周血和小肠粘膜组织内的水平变化;同时,应用阿苯达唑质脂体治疗自然感染细粒棘球蚴感染的绵羊,进一步评价细粒棘球蚴感染后对外周血及肠粘膜内Th1/Th2所介导的免疫反应的影响;此外,通过对肝细粒棘球蚴患者与正常人群中幽门螺杆菌感染率的进行病例对照研究,以期从肠道微生态角度来探讨,肠道微生态变化对细粒棘球蚴影响肠粘膜免疫屏障功能可能起到的作用。最终来研究,自然感染细粒棘球蚴对肠道粘膜免疫屏障可能发生的影响,为今后预防和治疗细粒棘球蚴感染提供可行性研究方向。方法:用B超初检和CT复检的方法,从新疆巴音布鲁克草原986只绵羊中,筛选出年龄在24-36月左右,自然感染细粒棘球蚴的雌性绵羊20只,按随机数字表法,分为两组,分别为使用阿苯达唑治疗组和细粒棘球蚴感染组,以及按同法筛选出未感染组绵羊10只,做为正常对照组。严格随机三盲方法,对上述三组实验对象进行治疗及饲养3月后,进行外周血采集、处死后小肠粘膜标本收集。用ELISA法对外周血中IL-6,IL-10及IFN-γ等免疫因子水平进行测定,分析细粒棘球感染及阿苯达唑质脂体治疗后外周血中免疫因子的变化;同时通过对小肠粘膜石蜡切片进行免疫组化染色,比较IL-6,IL-10及IFN-γ等免疫因子的阳性表达水平;此外,通过随机选取临床确诊感染细粒棘球蚴患者51例及正常健康体检人群30例成人做为对照组,使用化学发光法检测幽门螺杆菌感染情况,进一步探讨,肠道微生态对细粒棘球蚴作用于肠粘膜免疫屏障过程中的可能影响。结果:1)ELISA法对自然感染细粒棘球蚴绵羊外周血免疫因子IL-6、IL-10、IFN-γ进行测定提示:感染组上述3种免疫因子水平均高于对照组,但只有IL-10具有统计学意义(P0.001);免疫组化法对自然感染细粒棘球蚴绵羊肠粘膜内免疫因子IL-6、IL-10、IFN-γ进行测定提示:感染组仅有IFN-γ明显高于对照组,并有统计学意义(P0.005);2)阿苯达唑脂质体治疗干预后,ELISA法测定外周血Th2相关免疫因子IL-6,IL-10均有降低,但仅有IL-10具有统计学意义(P0.01);阿苯达唑脂质体治疗后,肠粘膜免疫组化结果示:肠粘膜内Th1相关免疫因子IFN-γ保持在较高水平,并具有统计学意义(P.01),肠粘膜内Th2相关免疫因子IL-10有所降低,但没有统计学意义(P=0.893);3)用化学发光法对51例细粒棘球感染患者进幽门螺杆菌抗体检测发现阳性率达74.51%,而对照组为50.00%,两者具有统计学差异(P=0.025)。结论:本研究利用自然感染细粒棘球蚴绵羊做为研究对象,更接近同样做为细粒棘球感染中间宿主的人类感染模式。研究发现,自然感染细粒棘球蚴绵羊外周血中免疫反应主要以Th2型为主,而肠粘膜免疫反应可能以Th1型为主;行阿苯达唑质脂体进行干预治疗后结果提示:阿苯达唑质脂体可增强机体体液及肠粘膜免疫屏障的Th1型免疫反应,对Th2类免疫因子有抑制作用;在对人细粒棘球蚴感染与幽门螺杆菌感染相关性病例对照研究结果提示:肠道微生态的变化可能对细粒棘球蚴感染后,肠粘膜免疫屏障功能改变具有影响,其中幽门螺杆菌可能与细粒棘球蚴存在协同感染可能,为进一步研究细粒棘球蚴如何影响肠粘膜屏障提供了一个可能的研究方向。
[Abstract]:Background: caused by Echinococcus larval hydatid disease is a zoonotic parasitic disease, high incidence in our country and around the world in the area of animal husbandry, the geographical distribution is very wide and has become a global public health problem. Human beings and some herbivorous animal (such as sheep) because eating eggs the discharge from the end host as its intermediate host, the eggs of the stomach, duodenum and small intestine in the final sojourn in the intestinal mucosa, the eggs hatched six larvae, through the intestinal mucosal barrier in the blood and reach the liver and other organs, great harm to the health of the body. The previous research found that Echinococcus granulosus has been able to long-term live in the body, it is through the type two Th1 and Th2 affect the host cellular immune response balance factor induced Th2 immune response of non protection become dominant, in favor of Echinococcus multilocularis in life Long. However, the intestinal mucosal immune barrier system is the largest lymphoid immune system, is the first barrier of Echinococcus granulosus into the body, the related research is rarely reported in its effect on intestinal mucosal immune barrier, through the research of natural infection of Echinococcus granulosus sheep as the object of animal research, a randomized controlled experiment, application enzyme linked immunosorbent assay (ELISA) and immunohistochemistry method, analysis of some immune factors related to Th1/Th2 (IL-6, IL-10 and IFN- gamma) levels in peripheral blood and intestinal mucosal tissues; at the same time, the application of albendazole liposome in treatment of natural infection of Echinococcus granulosus infection in sheep, immune the reaction further evaluation of Echinococcus granulosus infection after peripheral blood and intestinal mucosa mediated by Th1/Th2; in addition, the rate of Helicobacter pylori infection in patients with hepatic hydatid cyst and normal people. A case-control study was conducted, in order from the perspective of the intestinal microflora, intestinal mucosal barrier function of intestinal microflora changes of Echinococcus granulosus may play a role. To study the final, natural infection of Echinococcus granulosus may occur on intestinal mucosal immune barrier impact for future prevention and treatment of Echinococcus granulosus infection in the direction of the feasibility study provide. Methods: a re examination of the ultrasound examination and CT at the beginning of 986 sheep from Xinjiang Bayinbuluke grassland, screened at the age of 24-36 months, natural infection of Echinococcus granulosus in female 20 sheep were randomly divided into two groups, respectively using albendazole treatment group and Echinococcus granulosus the infection group, and according to the same method to filter out non infection group of 10 sheep, as normal control group. Three strictly random blind method, the three groups were treated by feeding and after March. Collect peripheral blood, after the death of intestinal mucosa were collected. Using ELISA method in peripheral blood IL-6, immune factors on levels of IL-10 and IFN- gamma were measured to analysis the changes of immune factor in peripheral blood of Echinococcus infection and albendazole liposome after treatment; at the same time through the intestinal mucosa paraffin sections immunohistochemical staining, IL-6, IL-10 and IFN- gamma serum expression level; in addition, the randomly selected clinical diagnosis of Echinococcus granulosus infection in 51 cases of patients and 30 cases of adult normal healthy people as the control group. The further study, the detection of Helicobacter pylori infection using chemiluminescence method, may affect the intestinal micro ecology of Echinococcus granulosus effect on intestinal mucosal barrier in the process. Results: 1) ELISA method on natural infection of Echinococcus granulosus sheep peripheral blood immune factors IL-6, IL-10, IFN- were determined by gamma tip: infection group The above 3 kinds of immune factors were higher than those in control group, but only IL-10 was statistically significant (P0.001); IL-10 immunohistochemical method on natural infection of Echinococcus granulosus sheep gut mucosal immune factors IL-6, IFN-, gamma determination tip: IFN- gamma group only significantly higher than the control group, with statistical significance (P0.005); 2) liposomal albendazole treatment after intervention, determination of peripheral blood Th2 related immune factors IL-6 ELISA, IL-10 had decreased, but only IL-10 was statistically significant (P0.01); albendazole liposome in the treatment of intestinal mucosa after immunohistochemical results are shown: in the intestinal mucosa of Th1 related immune factors IFN- gamma keep at a high level, and has statistical significance (P.01), in the intestinal mucosa of Th2 related immune factor IL-10 decreased, but not statistically significant (P=0.893); 3) by chemiluminescence method in 51 cases of Echinococcus infection in patients with Helicobacter pylori antibody detection The positive rate was 74.51%, and 50% in the control group, both had statistical difference (P=0.025). Conclusion: This study using the natural infection of Echinococcus granulosus sheep as research object, to do the same pattern as the human infection of Echinococcus infection of intermediate host. The study found that the natural infection of Echinococcus granulosus sheep immunity in peripheral blood the reaction mainly Th2 type and intestinal mucosal immune response may be dominated by Th1; for albendazole liposome intervention after treatment showed that albendazole liposome can enhance the humoral immune response to Th1 and intestinal mucosal immune barrier, inhibits Th2 immune factor; on Echinococcus granulosus infection with Helicobacter pylori infection related case-control study results suggested that the changes of intestinal microflora of Echinococcus granulosus infection, intestinal mucosal barrier function has the effect of changing, Helicobacter pylori may have synergistic infection with Echinococcus granulosus, which provides a possible direction for further research on how Echinococcus granulosus affects intestinal mucosal barrier.

【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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