日本血吸虫童虫细胞型免疫原的确认与免疫调节剂联用抗感染效果观察

发布时间：2018-06-05 17:29

本文选题：日本血吸虫 + 童虫细胞型免疫原　；参考：《中南大学》2008年硕士论文

【摘要】： 第一章Sj童虫细胞型与非细胞型免疫原诱生的保护性差异【目的】前期在小鼠模型中研究证明日本血吸虫(Sj)童虫活细胞可诱生抗攻击感染免疫力。本研究为证明其保护性效果是否与免疫原物理性状有关,比较童虫细胞型和非细胞型免疫原所诱生的保护性差异。【方法】用家兔经Sj尾蚴感染后第18d获得童虫,剪切法制备童虫细胞,细胞超声冻融制备非细胞抗原。动物实验:取50只单性清洁级昆明鼠随机分成对照(PBS)组、童虫细胞型抗原(SCA)免疫组和童虫细胞非细胞型抗原(SNCA)免疫组、童虫碎片抗原(SWFA)免疫组和童虫可溶性抗原(SWSA)免疫组。经小鼠腹股沟皮下肌肉接种免疫原3次,每次间隔2周。末次接种后第4周作攻击感染(30±1尾/鼠),感染后第45d观察虫荷、肝卵荷、肝虫卵肉芽肿大小。此外,用ELISA检测实验鼠血清中特异性抗体动态变化。【结果】PCR鉴定所用童虫细胞无兔源性成分。保护性指标比较显示:SCA组、SNCA组、SWFA组和SWSA组的减虫率分别为46.8%、35.3%、31.4%和8.0%;减卵率分别为:56.2%、38.2%、30.6%和20.2%;SWSA组未见明显减卵效果(P＞0.05)。SCA组小鼠肝虫卵肉芽肿显著小于其他4组(P＜0.05)。免疫学指标检测显示:SCA组、SNCA组、SWFA组和SWSA组各鼠血清中均可检测到抗Sj童虫抗体,其滴度与免疫次数呈正相关,在各组间差别不显著(P＞0.05)。【结论】进一步证明Sj童虫细胞型免疫原可诱导小鼠产生较理想的保护性效果,其诱生机制之一,与免疫原物理性状有关。第二章Sj童虫细胞型与非细胞型免疫原在小鼠注射部位的动态变化【目的】为证明Sj童虫细胞型免疫原诱生的保护性机制是否与免疫原在宿主体内缓慢释放有关,并观察细胞型抗原引起的病理学变化。【方法】昆明鼠设A,B两组:A组经大腿皮下肌肉注射10~7童虫细胞型抗原;B组注射非细胞抗原,注后分别在24hrs、3d、6d、9d和12d每组各处理1只小鼠,从注射部位定量获取组织,用Westernblot法和免疫组化鉴定比较两种类型抗原消失时间的动态差异;用组织病理学观察炎症变化动态。【结果】SDS-PAGE结果显示A、B两组各组织抗原泳道间的区带谱未见明显差异,Western-blot结果揭示,A组小鼠组织在细胞型抗原注射后12d仍可检测到血吸虫抗原持续存在75kDa蛋白条带,而B组非细胞型抗原在注后第3d消失。免疫组化检测抗原的结果与Western blot结果相符。A、B两组小鼠免疫部位的组织病理学观察显示,在注抗原后第1d未见炎性病变,从第3天开始肌肉间隙内出现大量炎性细胞浸润,其中A组小鼠的炎性病变持续至抗原注射后12d,但B组消失于抗原注后第12d已基本见不到炎性细胞浸润,A、B两组的炎性细胞均以中性粒细胞和淋巴细胞浸润为主。两组鼠的抗原注射部位肌细胞均未见异常病变。【结论】血吸虫童虫细胞型免疫原较其非细胞抗原在接种部位可维持更长时间,具抗原缓释作用,但引起炎症病变也较明显,推测细胞免疫原的这一特性可能是诱生高保护性免疫的重要机制之一。第三章Sj童虫细胞与免疫调节剂联用的保护性效果观察【目的】观察免疫调节剂可否进一步提高Sj童虫细胞诱生的免疫保护性效果。【方法】制备感染后第18d童虫细胞。PCR扩增鉴定血吸虫虫源性细胞的种属特异性并检测细胞有无宿主成分污染。实验动物分为8组。A组为PBS对照组;B组为童虫细胞免疫组;C组为IL-2注射组;D组为IL-2+童虫细胞免疫组;E组为IFN-γ注射组;F组为IFN-γ+童虫细胞免疫组;G组为香菇多糖注射组;F组为香菇多糖+童虫细胞免疫组。免疫调节剂每天注射一次,连续5d,免疫部位均为双侧腹股沟皮下肌肉注射,间隔2w 1次,共3次。于末次免疫后第4w采取单盲法对各组小鼠进行攻击感染(30尾/鼠)。感染后第35d剖鼠冲虫观察结果。保护性效果观察指标包括:计数虫荷及肝卵荷;免疫学观察指标包括:ELISA检测免疫前后不同时点鼠血清中特异性抗体的动态变化。【结果】经PCR鉴定证明血吸虫细胞免疫原无宿主成分污染。保护性效果显示:与A组比较,B组减虫率为38.4%,减雌率42.1%;C组、D组、E组、F组和G组和H组的减虫率分别为13.5%、36.1%和32.7%、46.2%、18.3%和33.7%;减雌率分别为-2.1%、21.1%、38.9%、40%、30.5%和40%。B组、C组、D组、E组、F组、G组和H组的LEPG减少率分别为59.8%、26.6%、52.1%、47.7%、57.7%、34%和53.7%;EPF减少率分别为48.6%、25.2%、44.4%、34.7%、50%、28.5%和42.3%。免疫调节剂与细胞联合免疫组与单用细胞组比,特异性抗体水平差异无统计学意义。【结论】免疫增强剂均未能显著提高童虫细胞诱生的免疫保护性效果,但单独注射IFN-γ有一定的抗日本血吸虫免疫保护性效果。
[Abstract]:Chapter one Sj protective differences between cell type and non cellular immunogen of larvae
[Objective] to prove that the living cells of Schistosoma japonicum (Sj) can induce immunity against attack infection in the mouse model. This study is to prove whether the protective effect is related to the physical properties of the immunogen and to compare the protective differences between the cell type and the non cellular immunogen. [Methods] the Sj cercariae of rabbits were used. The child worm was obtained at 18D after dyed, and the cells were prepared by shearing, and the cells were prepared by ultrasonic freezing and thawing. 50 single clean grade Kunming mice were randomly divided into the control group (PBS), the SCA immunization group and the cell non cell antigen (SNCA) immune group, the SWFA immunization group and the children's soluble immune group. SWSA immunization group. The immunogen was inoculated 3 times in the subcutaneous muscle of the mouse, each interval was 2 weeks. The last inoculation was fourth weeks after the last inoculation (30 + 1 tails / rats). After infection, the worm, liver and egg granuloma were observed in 45d. In addition, the dynamic changes of specific antibodies in the serum of the rats were detected by ELISA. [results] PCR identification No rabbit source components were used in children's cells. The protective indexes of SCA, SNCA, SWFA and SWSA were 46.8%, 35.3%, 31.4% and 8%, respectively: 56.2%, 38.2%, 30.6% and 20.2%, respectively, in group SWSA (P > 0.05), the liver egg granuloma in the.SCA group was significantly smaller than the other 4 groups (P < 0.05). Immunology Index detection showed that the anti Sj antibody was detected in the serum of SCA group, SNCA group, SWFA group and SWSA group, and the titer was positively correlated with the number of immune times, and there was no significant difference between each group (P > 0.05). [Conclusion] further proof that the cell type immunogen of Sj can induce a better protective effect and one of the inducing mechanisms. It is related to the physical properties of immunogen.
The second chapter is about the dynamic changes of Sj cell type and non cellular immunogen in injection site of mice.
[Objective] to prove whether the protective mechanism of immunogen induced by Sj cell type is related to the slow release of immunogen in the host and to observe the pathological changes caused by cell type antigen. [Methods] Kunming rats were set up A, B two groups: A group was injected subcutaneous muscle of the thigh to 10~7 cell type antigen of 10~7; group B was injected with non cell antigen and injected after injection. Don't treat 1 mice in each group of 24hrs, 3D, 6D, 9D and 12D, obtain tissue from the injection site, compare the dynamic difference between the two types of antigen disappearance time by Westernblot and immunohistochemistry, and observe the changes of the inflammation by histopathology. [results] SDS-PAGE results show that the band spectrum of A, B two groups of tissue antigens in the B two groups The Western-blot results revealed that 12D in group A mice could still detect the persistence of 75kDa protein bands in 12D after injection of cell antigen, while the non cellular antigens in B group disappeared in post injection 3D. The results of immunohistochemical detection of antigen were in conformity with the results of Western blot, and the histopathology of the immune parts of the mice of B two group was histopathological. The observation showed that there was no inflammatory lesion in 1D after the injection of antigen, and a large number of inflammatory cells began to appear in the intermuscular space from third days. The inflammatory lesions in the A Group continued to 12D after the antigen injection, but the B group disappeared after the antigen injection at 12D, and the inflammatory cells in the group of A and B two were all neutrophils and lymph nodes. There was no abnormal lesion in the myocytes of the two groups. [Conclusion] the cell type immunogen of Schistosoma japonicum can maintain longer than its non cellular antigen at the inoculation site, and has the effect of antigen release, but the inflammatory disease is also more obvious. It is presumed that this characteristic of fine cell immunogen may be the high protection of the inducer. One of the important mechanisms of sexual immunity.
The third chapter is about the protective effect of Sj combined with immunomodulator.
[Objective] to observe whether the immunomodulator could further improve the protective effect of Sj on the cells induced by Sj. [Methods] the species specificity of.PCR amplification and identification of the source cells of Schistosoma japonicum after infection was prepared and detected by the host cell. The experimental animals were divided into 8 groups of.A groups as the PBS control group and the B group as a child. The cell immune group, group C was IL-2 injection group, group D was IL-2+ cell immunization group, E group was IFN- gamma injection group, F group was IFN- y + worm cell immunization group, G group was letinous edodes polysaccharide injection group, and F group was letinous edodes polysaccharide + worm cell immune group. 2W 1 times, a total of 3 times. After the last immunization, a single blind method was used to attack the mice in each group (30 tail / rat). The observation results of the 35d caesarean section after infection were observed. The protective effect observation included counting the worm and liver eggs. The immunological observation index included: ELISA was used to detect the activity of the specific antibody in the serum of the mice before and after the immunization. [results] PCR identification showed that Schistosoma cells had no host component contamination. The protective effect showed that compared with group A, the rate of worm reduction in group B was 38.4%, and the female rate was 42.1%; the rate of worm reduction in group C, D, E, F, G and H were 13.5%, 36.1% and 32.7%, 46.2%, 18.3% and 33.7%, respectively, and the reduction rate was -2.1%, 21.1%, 38.9%, 40%, 30.5%, respectively. The LEPG reduction rates of group 40%.B, group C, group D, E, F, G, and H were 59.8%, 26.6%, 52.1%, 47.7%, 57.7%, 34% and 53.7%, and the rate of EPF reduction was 48.6%, 25.2%, 44.4%, 34.7%, 50%, 28.5% and 42.3%. immunomodulators compared with the single cell group, and the specific antibody level was not statistically significant. [Conclusion] immunization Enhancers did not significantly enhance the immune protective effect of larvae, but IFN- gamma alone had some protective effects against Schistosoma japonicum.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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