日本血吸虫UV致弱尾蚴诱导不同品系小鼠的免疫保护作用及其相关机理的初步研究

发布时间：2018-07-27 20:03

【摘要】： 日本血吸虫病仍然是严重危害我国人民身体健康的寄生虫病。2003年全国调查资料显示，我国共有现症患者80余万，受威胁人口达1亿。目前，血吸虫病的防治工作强调以化疗为主，结合钉螺控制、健康教育等手段来降低血吸虫病的危害。虽然，，化疗起到了很好的治疗作用，但仍存在一些问题，如：化疗不能控制再感染，无法彻底阻断疾病的传播，长期使用吡喹酮可能导致抗药性的产生等。鉴于疫苗的研究和使用在世界范围内为人类预防和消灭许多传染性疾病起到了非常重要的作用，因此，研究血吸虫病疫苗，通过“短效”的化疗作用及疫苗接种后产生的“长效”免疫预防作用相结合来控制血吸虫病，成为上个世纪80年代以来全世界科学家的共识。血吸虫病疫苗的研究历时70余年，大体上经历了死疫苗、减毒活疫苗、基因工程疫苗、核酸疫苗研究阶段。迄今为止，在动物实验中，以辐照致弱尾蚴或童虫为免疫原的预防效果最佳。当然，应用辐照致弱尾蚴直接作为疫苗，存在着抗原材料有限、具有潜在感染的危险及可能引发有限病变的伦理问题等。但是，利用致弱尾蚴免疫模型揭示疫苗诱导保护力的机制，无疑将为最终获得行之有效的疫苗提供重要的理论基础。目前，关于辐照致弱尾蚴生物学的研究比较有限，包括其活力、外部形态、内部显微结构及抗原成分等，其诱导宿主产生免疫保护力的分子机制可能与致弱尾蚴进入宿主后的抗原递呈不同于正常尾蚴有关，包括辐照所致的尾蚴移行延迟、抗原成分改变(隐蔽抗原的暴露)及失去了正常尾蚴中下调免疫应答的特定蛋白等有关。因此，观察辐照致弱尾蚴的生物学改变，是研究其诱导相关的免疫保护机理的实验基础。值得注意的是，国内外对辐照致弱尾蚴的相关免疫学研究多集中在曼氏血吸虫，其在许多品系小鼠中可诱导60％～80％的保护力，γ射线致弱尾蚴免疫非人灵长类动物后可诱导产生大于50％的保护力。与曼氏血吸虫相比，对日本血吸虫辐照致弱尾蚴的研究起步较晚，多在借鉴曼氏血吸虫相关研究经验的基础上开展。而现有资料表明，日本血吸虫辐照致弱尾蚴在小动物(特别是小鼠)中诱导产生的保护力水平常不稳定，目前尚缺乏公认的能对其产生稳定高保护力的小鼠模型。本实验室曾在昆明鼠、DBA及C57BL／6小鼠品系进行小规模的UV(Ultra-Violet，紫外线)致弱尾蚴保护力实验，希望构建日本血吸虫致弱尾蚴诱导保护力的小鼠模型，虽然抗体检测提示致弱尾蚴免疫诱导了高于正常尾蚴感染的特异性体液免疫应答，但是均未获得预期的保护力效果。本课题进一步深入地在多品系的小鼠进行了免疫保护实验，并观察比较了各实验组DBA小鼠的Th1／Th2细胞免疫应答特征，力图探讨小鼠的品系、免疫应答模式与对日本血吸虫感染的保护力之间的关系。本研究首先观察了UV致弱尾蚴和正常尾蚴活力，并利用扫描电镜、透射电镜及免疫电镜系统地观察日本血吸虫UV致弱尾蚴在外部形态、内部显微结构、抗原成分及其定位与正常尾蚴的异同。其次，选取昆明鼠作为观察对象，观察了不同UV照射剂量、紫外光源等因素对免疫保护力的影响。在此基础上，我们又比较了不同鼠系(昆明鼠、DBA、BALB／c)经免疫后诱导的保护力；同时，对DBA小鼠的细胞免疫应答进行了动态观察。旨在继续探索可用于日本血吸虫UV致弱尾蚴免疫保护机制研究的小鼠模型，并探讨与日本血吸虫UV致弱尾蚴保护力有关的免疫应答机制。本研究获得结果如下： 1．尾蚴活力观察：经200、445(UV照射40秒)和800μw／cm~2的UV照射1min，尾蚴的死亡率分别为：1.78％、1.73％和2.09％。经三种UV强度照射的尾蚴与未经照射的尾蚴的活力间无显著性差异(P＞0.05)。 2．尾蚴体表及内部结构的电镜观察：UV致弱尾蚴与正常尾蚴相比，①其体部高度收缩，具有明显皱褶，尾部分叉明显肿胀，体部向内凹陷，且体棘缺损，边缘粗糙；②肌纤维层变薄，有少许断裂，线粒体未见明显的嵴状结构；③UV致弱尾蚴切片上的胶体金颗粒少于正常尾蚴切片，特别是肌纤维处，且UV致弱尾蚴切片上的胶体金颗粒在多部位有聚集分布现象。由此可能提示，UV照射损伤了尾蚴的体表、肌肉、重要代谢器官的结构，尾蚴抗原成分也可能发生了变化。 3．昆明鼠、DBA及BALB／c小鼠为对日本血吸虫UV致弱尾蚴低应答的小鼠品系。昆明鼠经不同UV强度、不同紫外源照射的日本血吸虫尾蚴免疫后的保护力为-1.0～23.75％；昆明鼠、DBA、BALB／c小鼠经100条／鼠的免疫剂量、免疫与攻击间隔4周，致弱尾蚴所诱导的保护力均较低(8.13％～26.9％)。与本实验室前期保护力实验的结果一致，提示昆明鼠、DBA、BALB／c小鼠不适宜作为日本血吸虫UV致弱尾蚴免疫保护力研究的动物模型。 4．观察DBA鼠在日本血吸虫UV致弱尾蚴免疫组、正常尾蚴感染组及先免疫后攻击组之间血清中IL-4、IL-10、IFN-γ及IL-12p40的水平变化。结果显示，致弱尾蚴免疫组小鼠血清中IL-12p40在免疫后第3周降到最低水平，但是总体上细胞因子在0～4周并未产生明显变化；与感染组和先免疫后攻击组相比，细胞因子总体水平较低，尽管IL-10及IFN-γ在3组之间的各个时间点并无显著差异(P＞0.05)，且免疫组IL-12p40的水平在第1周和第2周时又分别高于感染组和先免疫后攻击组(P＜0.05)；对比感染组与先免疫后攻击组细胞因子水平可以看出，两组之间4种细胞因子的水平并无显著差异。可见，宿主在UV致弱尾蚴免疫后未能产生有效的细胞免疫应答，这可能是日本血吸虫致弱尾蚴免疫在该品系小鼠不能产生免疫保护作用的主要原因。本研究结果进一步丰富了日本血吸虫UV致弱尾蚴保护性免疫机制的研究，为构建适宜的UV致弱尾蚴免疫保护力的动物模型提供了重要的基础资料。
[Abstract]:Schistosomiasis is still a parasitic disease that seriously endangers the health of the people of our country in the national survey of.2003, which shows that there are more than 80 million patients with present symptoms and 100 million of the population threatened.
At present, the prevention and control of schistosomiasis emphasizes chemotherapy mainly, combined with Oncomelania control, health education and other means to reduce the harm of schistosomiasis. Although chemotherapy has played a very good therapeutic role, there are still some problems, such as: chemotherapy can not control re infection, can not block the spread of the disease thoroughly, and the long-term use of praziquantel may lead to As the research and use of vaccines play a very important role in the prevention and elimination of many infectious diseases worldwide, the study of schistosomiasis vaccines can be used to control schistosomiasis through the combination of "short effective" chemotherapy and the "long effect" immunization after vaccination. Become the consensus of scientists around the world since the 80s of last century.
The study of schistosomiasis vaccine lasted for more than 70 years. It has experienced dead vaccine, live attenuated vaccine, genetic engineering vaccine, and nucleic acid vaccine research stage. So far, in animal experiments, the prevention effect of irradiated cercariae or children as immunogens is the best in animal experiments. Of course, the use of irradiated cercariae is a direct vaccine and there is an antigen material. Limited, it has the risk of potential infection and the ethical problems that may lead to the limited disease. However, the mechanism of using the immune model of the cercariae to reveal the protective ability of the vaccine will undoubtedly provide an important theoretical basis for the eventual effective vaccine.
At present, the study on the biology of irradiated cercariae is limited, including its vitality, external morphology, internal microstructure and antigen components. The molecular mechanism of inducing immune protection by the host may be different from the normal cercariae of the antigen after the entry of the weak cercariae to the host, including the delayed migration of cercariae caused by irradiation, The changes in the antigen composition (the exposure of the hidden antigen) and the loss of the specific proteins that reduce the immune response in the normal cercariae are related. Therefore, the observation of the biological changes of the irradiated cercariae is the experimental basis for the study of the related mechanism of immune protection.
It is worth noting that the related immunological studies of irradiated cercariae at home and abroad are mostly concentrated in the Schistosoma mansoni, which can induce 60% to 80% of the protection in many strains of mice, and the weak cercariae of gamma rays can induce more than 50% of the protective power of the nonhuman primates. Compared with Schistosoma mansoni, the radiation of Schistosoma japonicum is compared with the Schistosoma mansoni. The research on the weak cercariae started relatively late and has been carried out on the basis of the related research experience of Schistosoma mansoni. The existing data show that the protective ability of the weak cercariae induced by Schistosoma japonicum in small animals (especially in mice) is often unstable. At present, it is still lacking in the recognized mice that can produce stable and high protective ability to it. Model. A small scale UV (Ultra-Violet, ultraviolet) induced weak cercariae protection test was carried out in Kunming mice, DBA and C57BL / 6 mice, hoping to construct a mouse model of the weak cercariae induced by Schistosoma japonicum, although the antibody detection suggested that the immunization of the weak cercariae induced the specific body fluid higher than the normal cercariae infection. The immune response was not obtained, but the immune response of the mice in the multi strain system was further studied. The immune response characteristics of the Th1 / Th2 cells of the DBA mice in the experimental groups were observed and compared. The model of mice, the immune answer pattern and the protection of the infection of Schistosoma japonicum were explored. The relationship.
In this study, the activity of UV induced cercariae and normal cercariae was observed. Scanning electron microscopy, transmission electron microscopy and immunoelectron microscopy were used to observe the external morphology, internal microstructure, antigen composition and the similarities and differences between the antigen and the normal cercariae of the weak cercariae of Schistosoma japonicum UV.
Secondly, Kunming mice were selected as observation objects, and the effects of different UV irradiation doses and ultraviolet light sources on immune protection were observed. On this basis, we also compared the protective ability of different mice (Kunming mice, DBA, BALB / C) after immunization. At the same time, the cellular immune response of DBA mice was dynamically observed. To explore a mouse model which can be used to study the immune protection mechanism of cercariae induced by Schistosoma japonicum UV, and to explore the immune response mechanism related to the weak cercariae induced by Schistosoma japonicum UV.
The results of this study are as follows:
1. cercariae vigour observation: the mortality of cercariae was 1.78%, 1.73% and 2.09%. were 1.78%, 1.73% and 2.09%. were irradiated by three UV intensities, and there was no significant difference between the activity of three UV intensity and the vitality of the unirradiated cercariae (P > 0.05). The mortality of cercariae was 200445 (UV irradiated for 40 seconds) and 800 mu / cm~2.
2. cercariae body surface and the internal structure of the electron microscope observation: UV induced weak cercariae and normal cercariae compared with the normal cercariae, the body part is highly contracted, with obvious wrinkles, the tail bifurcation obviously swollen, the body part is inward, and the body spines defect, the edge is rough; the muscle fiber layer is thinner, there are a few breakages and the mitochondria have no obvious ridge structure; (3) UV caused the weak cercariae The colloid gold particles on the slice were less than the normal cercariae slices, especially the muscle fibers, and the colloidal gold particles on the UV induced cercariae slices were aggregated in many parts. Thus, it may be suggested that UV irradiation damage the body surface, muscle, structure of important metabolic organs and the antigen composition of cercariae in the cercariae.
3. Kunming mice, DBA and BALB / c mice were mice with low response to Schistosoma japonicum UV induced weak cercariae. The protective power of Kunming mice after immunization with different UV intensities and different ultraviolet irradiated cercariae of Schistosoma japonicum was -1.0 ~ 23.75%; Kunming mice, DBA, BALB / c mice were immunized with 100 mice and immune and attack intervals for 4 weeks. The protections induced by cercariae were all low (8.13% ~ 26.9%). It was consistent with the results of previous experiments in our laboratory, suggesting that the mice of Kunming mice, DBA, BALB / c mice were not suitable for the animal model of the study of the immunoprotective ability of the weak cercariae caused by UV of Schistosoma japonicum.
4. the changes in serum levels of IL-4, IL-10, IFN- gamma and IL-12p40 in the serum of DBA mice induced by UV of Schistosoma japonicum, the normal cercariae infection group and the pre immunized attack group were observed. The results showed that the IL-12p40 in the mice of the weak cercariae immune group dropped to the lowest level in the third weeks after the immunization, but the overall cytokine was 0~4 weeks. Compared with the infection group and the first immunization group, the overall level of cytokine was low, although there was no significant difference between IL-10 and IFN- gamma (P > 0.05) at each time point between the 3 groups, and the level of IL-12p40 in the immune group was higher than that of the infected group and the pre immunized attack group at first weeks and second weeks (P < 0.05); The level of cytokine in the infection group and the pre immunized attack group showed that there was no significant difference in the level of 4 cytokines between the two groups. It could be seen that the host could not produce an effective cellular immune response after the immunization of the weak cercariae caused by UV, which may be the main reason for the immune protection of the weak cercariae induced by Schistosoma japonicum in the strain mice. For reasons.
The results of this study further enrich the protective immune mechanism of Schistosoma japonicum UV induced cercariae, and provide important basic information for the establishment of an appropriate animal model for the immune protection of UV induced cercariae.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R383

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