【摘要】： 日本血吸蟲病仍然是嚴重危害我國人民身體健康的寄生蟲病。2003年全國調(diào)查資料顯示，我國共有現(xiàn)癥患者80余萬，受威脅人口達1億。 目前，血吸蟲病的防治工作強調(diào)以化療為主，結(jié)合釘螺控制、健康教育等手段來降低血吸蟲病的危害。雖然，，化療起到了很好的治療作用，但仍存在一些問題，如：化療不能控制再感染，無法徹底阻斷疾病的傳播，長期使用吡喹酮可能導(dǎo)致抗藥性的產(chǎn)生等。鑒于疫苗的研究和使用在世界范圍內(nèi)為人類預(yù)防和消滅許多傳染性疾病起到了非常重要的作用，因此，研究血吸蟲病疫苗，通過“短效”的化療作用及疫苗接種后產(chǎn)生的“長效”免疫預(yù)防作用相結(jié)合來控制血吸蟲病，成為上個世紀80年代以來全世界科學(xué)家的共識。 血吸蟲病疫苗的研究歷時70余年，大體上經(jīng)歷了死疫苗、減毒活疫苗、基因工程疫苗、核酸疫苗研究階段。迄今為止，在動物實驗中，以輻照致弱尾蚴或童蟲為免疫原的預(yù)防效果最佳。當然，應(yīng)用輻照致弱尾蚴直接作為疫苗，存在著抗原材料有限、具有潛在感染的危險及可能引發(fā)有限病變的倫理問題等。但是，利用致弱尾蚴免疫模型揭示疫苗誘導(dǎo)保護力的機制，無疑將為最終獲得行之有效的疫苗提供重要的理論基礎(chǔ)。 目前，關(guān)于輻照致弱尾蚴生物學(xué)的研究比較有限，包括其活力、外部形態(tài)、內(nèi)部顯微結(jié)構(gòu)及抗原成分等，其誘導(dǎo)宿主產(chǎn)生免疫保護力的分子機制可能與致弱尾蚴進入宿主后的抗原遞呈不同于正常尾蚴有關(guān)，包括輻照所致的尾蚴移行延遲、抗原成分改變(隱蔽抗原的暴露)及失去了正常尾蚴中下調(diào)免疫應(yīng)答的特定蛋白等有關(guān)。因此，觀察輻照致弱尾蚴的生物學(xué)改變，是研究其誘導(dǎo)相關(guān)的免疫保護機理的實驗基礎(chǔ)。 值得注意的是，國內(nèi)外對輻照致弱尾蚴的相關(guān)免疫學(xué)研究多集中在曼氏血吸蟲，其在許多品系小鼠中可誘導(dǎo)60％～80％的保護力，γ射線致弱尾蚴免疫非人靈長類動物后可誘導(dǎo)產(chǎn)生大于50％的保護力。與曼氏血吸蟲相比，對日本血吸蟲輻照致弱尾蚴的研究起步較晚，多在借鑒曼氏血吸蟲相關(guān)研究經(jīng)驗的基礎(chǔ)上開展。而現(xiàn)有資料表明，日本血吸蟲輻照致弱尾蚴在小動物(特別是小鼠)中誘導(dǎo)產(chǎn)生的保護力水平常不穩(wěn)定，目前尚缺乏公認的能對其產(chǎn)生穩(wěn)定高保護力的小鼠模型。本實驗室曾在昆明鼠、DBA及C57BL／6小鼠品系進行小規(guī)模的UV(Ultra-Violet，紫外線)致弱尾蚴保護力實驗，希望構(gòu)建日本血吸蟲致弱尾蚴誘導(dǎo)保護力的小鼠模型，雖然抗體檢測提示致弱尾蚴免疫誘導(dǎo)了高于正常尾蚴感染的特異性體液免疫應(yīng)答，但是均未獲得預(yù)期的保護力效果。本課題進一步深入地在多品系的小鼠進行了免疫保護實驗，并觀察比較了各實驗組DBA小鼠的Th1／Th2細胞免疫應(yīng)答特征，力圖探討小鼠的品系、免疫應(yīng)答模式與對日本血吸蟲感染的保護力之間的關(guān)系。 本研究首先觀察了UV致弱尾蚴和正常尾蚴活力，并利用掃描電鏡、透射電鏡及免疫電鏡系統(tǒng)地觀察日本血吸蟲UV致弱尾蚴在外部形態(tài)、內(nèi)部顯微結(jié)構(gòu)、抗原成分及其定位與正常尾蚴的異同。 其次，選取昆明鼠作為觀察對象，觀察了不同UV照射劑量、紫外光源等因素對免疫保護力的影響。在此基礎(chǔ)上，我們又比較了不同鼠系(昆明鼠、DBA、BALB／c)經(jīng)免疫后誘導(dǎo)的保護力；同時，對DBA小鼠的細胞免疫應(yīng)答進行了動態(tài)觀察。旨在繼續(xù)探索可用于日本血吸蟲UV致弱尾蚴免疫保護機制研究的小鼠模型，并探討與日本血吸蟲UV致弱尾蚴保護力有關(guān)的免疫應(yīng)答機制。 本研究獲得結(jié)果如下： 1．尾蚴活力觀察：經(jīng)200、445(UV照射40秒)和800μw／cm~2的UV照射1min，尾蚴的死亡率分別為：1.78％、1.73％和2.09％。經(jīng)三種UV強度照射的尾蚴與未經(jīng)照射的尾蚴的活力間無顯著性差異(P＞0.05)。 2．尾蚴體表及內(nèi)部結(jié)構(gòu)的電鏡觀察：UV致弱尾蚴與正常尾蚴相比，①其體部高度收縮，具有明顯皺褶，尾部分叉明顯腫脹，體部向內(nèi)凹陷，且體棘缺損，邊緣粗糙；②肌纖維層變薄，有少許斷裂，線粒體未見明顯的嵴狀結(jié)構(gòu)；③UV致弱尾蚴切片上的膠體金顆粒少于正常尾蚴切片，特別是肌纖維處，且UV致弱尾蚴切片上的膠體金顆粒在多部位有聚集分布現(xiàn)象。由此可能提示，UV照射損傷了尾蚴的體表、肌肉、重要代謝器官的結(jié)構(gòu)，尾蚴抗原成分也可能發(fā)生了變化。 3．昆明鼠、DBA及BALB／c小鼠為對日本血吸蟲UV致弱尾蚴低應(yīng)答的小鼠品系。昆明鼠經(jīng)不同UV強度、不同紫外源照射的日本血吸蟲尾蚴免疫后的保護力為-1.0～23.75％；昆明鼠、DBA、BALB／c小鼠經(jīng)100條／鼠的免疫劑量、免疫與攻擊間隔4周，致弱尾蚴所誘導(dǎo)的保護力均較低(8.13％～26.9％)。與本實驗室前期保護力實驗的結(jié)果一致，提示昆明鼠、DBA、BALB／c小鼠不適宜作為日本血吸蟲UV致弱尾蚴免疫保護力研究的動物模型。 4．觀察DBA鼠在日本血吸蟲UV致弱尾蚴免疫組、正常尾蚴感染組及先免疫后攻擊組之間血清中IL-4、IL-10、IFN-γ及IL-12p40的水平變化。結(jié)果顯示，致弱尾蚴免疫組小鼠血清中IL-12p40在免疫后第3周降到最低水平，但是總體上細胞因子在0～4周并未產(chǎn)生明顯變化；與感染組和先免疫后攻擊組相比，細胞因子總體水平較低，盡管IL-10及IFN-γ在3組之間的各個時間點并無顯著差異(P＞0.05)，且免疫組IL-12p40的水平在第1周和第2周時又分別高于感染組和先免疫后攻擊組(P＜0.05)；對比感染組與先免疫后攻擊組細胞因子水平可以看出，兩組之間4種細胞因子的水平并無顯著差異。可見，宿主在UV致弱尾蚴免疫后未能產(chǎn)生有效的細胞免疫應(yīng)答，這可能是日本血吸蟲致弱尾蚴免疫在該品系小鼠不能產(chǎn)生免疫保護作用的主要原因。 本研究結(jié)果進一步豐富了日本血吸蟲UV致弱尾蚴保護性免疫機制的研究，為構(gòu)建適宜的UV致弱尾蚴免疫保護力的動物模型提供了重要的基礎(chǔ)資料。
[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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R383

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6 麗妮;愛克發(fā)推出新的高速UV固化噴墨印刷機[N];中國包裝報;2009年

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10 楊璐;滿洲里局投入使用UV——C光學(xué)共聚焦顯微圖像系統(tǒng)[N];中國國門時報;2010年

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1 鄭會民;日本血吸蟲感染對宿主輔助性T細胞反應(yīng)的影響研究[D];復(fù)旦大學(xué);2011年

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