旋毛虫成虫丝氨酸蛋白酶对宿主免疫细胞影响的初步研究

发布时间：2018-02-28 16:22

本文关键词： 旋毛虫成虫丝氨酸蛋白酶免疫细胞细胞毒性增殖性应答　出处：《吉林大学》2012年硕士论文　论文类型：学位论文

【摘要】：旋毛虫病是由于人和动物生食或半生食含有旋毛虫感染性L1幼虫的肉或肉制品而引发的一种人兽共患病。本病致死率虽然不高（1-2%），仅重度感染才能造成严重的心肌及大脑损伤而导致死亡，但是在其肠道侵袭期通过免疫调控抑制宿主免疫力，同时干扰了各种细菌、病毒以及其他寄生虫疫苗的免疫预防效果，大大提高了其他病原体入侵宿主的可能性，对人类身体健康构成了巨大的威胁。但是目前对于这些旋毛虫调节宿主免疫反应的毒力分子不确定性及其作用机制尚不清楚导致了有关旋毛虫病的预防制剂的研发始终没有突破性的进展。本实验室通过构建了旋毛虫3日龄成虫cDNA文库，，并利用免疫学筛选获得了高丰度和强抗原性的丝氨酸蛋白酶基因Zh68。寄生虫丝氨酸蛋白酶在宿主与寄生虫之间互相作用过程（如入侵宿主组织、寄生虫营养的摄取和逃避宿主免疫应答）中发挥了重要作用，甚至有研究表明丝氨酸蛋白酶可作为多种寄生虫感染性疾病的诊断标志。本实验通过对旋毛虫成虫肠道感染期宿主多种免疫细胞的动态变化监测来初步剖析宿主免疫抑制的变化规律。首先将500条L1幼虫口服感染昆明小鼠，发现在感染后第1天小鼠肠道成虫回收率为65%，直至感染后第17天小鼠肠道成虫完全排出；感染后第5天到第9天是成虫稳定的肠道感染期，此时雌虫大量释放新生幼虫，并于第7天达到高峰。随后的流式细胞术检测结果显示感染后第7天CD4~+T淋巴细胞数量大量减少，同时T淋巴细胞对刀豆素A（Con A）的增殖性应答能力降低。本实验室在前期构建的新生幼虫cDNA文库中也筛选到了特异性丝氨酸蛋白酶基因T668，因此很有可能是成虫丝氨酸蛋白酶Zh68和新生幼虫特异性丝氨酸蛋白酶T668共同导致了这一结果的产生。在旋毛虫感染后的前7天，T淋巴细胞增多，以CD8~+T淋巴细胞为主，导致CD4~+/CD8~+比值下降，表明小鼠免疫力降低。从旋毛虫感染后第11天开始，CD4~+T细胞明显增多，小鼠免疫力逐步恢复。同时发现在旋毛虫肠道感染前期（感染前7天），小鼠巨噬细胞数量急剧下降，相对于LPS的刺激，B淋巴细胞对旋毛虫抗原无增殖性应答；从感染后第11天开始巨噬细胞数量恢复，B淋巴细胞数量开始上升。鉴于旋毛虫肠道感染前期小鼠巨噬细胞急剧性变化，我们进一步通过体外实验研究旋毛虫成虫排泄分泌产物（ES产物）中的丝氨酸蛋白酶对巨噬细胞S774A.1的作用机制。CCK-8的实验结果表明成虫ES产物以剂量依赖性和时间依赖性的方式对S774A.1巨噬细胞产生毒性作用，但用抗成虫丝氨酸蛋白酶Zh68的抗体封闭ES产物之后，这一毒性作用大大减少；通过Annexin V/PI双标记流式细胞术检测结果表明成虫丝氨酸蛋白酶Zh68可以诱导巨噬细胞S774A.1凋亡。目前有关旋毛虫免疫逃逸相关基因的研究薄弱，研究旋毛虫丝氨酸蛋白酶在虫体侵袭及寄生过程中的功能及作用靶标、将为阐明旋毛虫侵袭过程中的免疫逃逸机制以及新型的抗旋毛虫及相关寄生虫药物研发打下结实基础。
[Abstract]:Trichinellosis is a zoonosis caused by human and animal eating raw or undercooked food containing L1 larvae infected with Trichinella spiralis meat or meat products. The prevalence of this disease mortality rate is not high (1-2%), only severe infections can cause serious heart and brain damage and lead to death, but is in theintestinal phase through the inhibition of immune regulation of host immunity, and the interference of a variety of bacteria, viruses and other parasites vaccine immune prevention effect, greatly improving the possibility of other pathogens invade the host, poses a great threat to human health. But at present for the virulence genes of Trichinella spiralis modulate the host immune response uncertainty and its mechanism of action is not clear cause the research on the prevention of trichinosis has not a breakthrough. The laboratory was constructed by Trichinella spiralis 3 days old adult cDNA library, And the use of immunological screening to obtain the high abundance and high antigenicity of the serine protease gene Zh68. parasites between serine proteases in the host parasite interaction process (such as the invasion of host tissues, the uptake of nutrients of parasites and evade the host immune response) play an important role, and even research indicates that serine protease can be used for diagnosis of infectious diseases of various parasites mark.
Through this experiment, dynamic monitoring of host immune cells of adult Trichinella spiralis intestinal infection to the preliminary analysis of changes of host immune suppression. The 500 L1 larvae of oral infection in Kunming mice, found in first days after infection of mice intestinal adult worm recovery rate is 65%, until seventeenth days after infection of mouse intestinal adult infection completely discharged; after fifth to ninth days is a stable adult intestinal infection period, the release of a large number of female newborn larvae, peaked at seventh day. The results of flow cytometry showed that seventh days after infection the number of lymph cells reduce subsequent CD4~+T, while T lymphocytes to concanavalin A (Con A) reduced the proliferative response ability of newborn larvae cDNA Library in our laboratory previously constructed also screened specific serine protease gene T668, so it's possible that the adult serine protease Zh6 8 newborn larvae and specific serine protease T668 leads to the results. In the first 7 days after infection with Trichinella spiralis, T lymphocytes increased to CD8~+T lymphocytes, resulting in the decrease of the ratio of CD4~+/CD8~+, showed that the immune system of mice decreased. From the eleventh day after infection with Trichinella spiralis, CD4~+T cells significantly increased the immunity of mice gradually restored. At the same time found in the early stage of intestinal infection of Trichinella spiralis (7 days before infection), a sharp decline in the number of macrophages of mice, compared with LPS stimulation, B lymphocyte proliferative response to antigen of Trichinella spiralis infection after eleventh days; the number of macrophages began recovery, the number of B lymphocytes began to rise.
In view of early intestinal infection of Trichinella spiralis in mouse macrophages change dramatically, we further product in vitro study of adult Trichinella spiralis excretory secretory (ES product) mechanism of.CCK-8 on macrophage S774A.1 serine protease in experimental results show that the adult ES product in a dose-dependent and time-dependent manner have toxic effects on S774A.1 macrophages, but after with the antibody against adult serine protease Zh68 closed ES products, which greatly reduce the toxicity; by Annexin V/PI double labeled flow cytometry results showed that adult serine protease Zh68 can induce apoptosis of S774A.1 macrophages.
At present the researches on immune escape related gene of Trichinella spiralis is weak, function and role in parasite invasion and parasitic in the process of target of Trichinella spiralis serine protease, will escape mechanism and new anti Trichinella parasites and lay a solid foundation for drug development in the process of invasion of Trichinella spiralis immune to clarify.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

【共引文献】