【作者】 张美娟；

【导师】 吴观陵；

【作者基本信息】 南京医科大学 ， 病原生物学， 2009， 博士

【摘要】 虽经半个多世纪的不懈抗争,日本血吸虫病(schistosomiasis japonica)迄今仍然是我国主要公共卫生问题之一。近年来,采取的以传染源控制为主的综合防治策略,将重点置于对构成我国现有疫区主要传染源--牛的控制上,似乎已经显示出较好的防治效果,但基本上没脱离以传播生态学为原理的思维。防治实践表明,依据疾病传播生态学原理设计的综合防治措施虽然有效,但效果难以巩固,极易受到自然生态环境和防治投入力度变化的挑战。鉴于疫苗在许多传染病控制中无可比拟的作用,试图发展血吸虫疫苗,获取化疗“短效”作用与疫苗接种诱导较“长效”的免疫预防作用相结合的双重效益来解决血吸虫病控制的期盼是近20-30年来世界各国有关科学家为之奋斗的目标之一,也是WHO/TDR自上世纪80年代以来在世界范围内协调血吸虫病疫苗研制努力的初衷。关于血吸虫病疫苗发展及相关的血吸虫病免疫学研究,大多集中在曼氏血吸虫病(schistosomiasis mansoni),日本血吸虫病疫苗发展研究还远滞后于曼氏血吸虫病相关研究,尤其是相关的免疫学基础研究更为薄弱。因此在努力研究和开发日本血吸虫病疫苗的过程中,不仅要重视现代生物学技术的进步所带来的机遇,而且更重要的是要回归到血吸虫感染免疫学的基础研究,以寻觅血吸虫病疫苗发展的合理策略和途径。在对曼氏血吸虫病疫苗进行研究的过程中,应用效应细胞、效应分子的抗体拮抗及基因缺陷小鼠模型等对辐照致弱(RA)尾蚴疫苗进行深入分析,发现对血吸虫病的保护性免疫主要依赖于抗原特异性的CD4+ Th细胞应答及体液免疫应答。这些关于保护性免疫的研究均集中于获得性免疫应答的效应端事件,而对天然免疫应答的启动端事件则涉及较少。迄今为止,在曼氏血吸虫感染中有关天然免疫应答的研究多集中在虫卵抗原,阐明为何在产卵之后宿主呈现Th2型优势应答。这些基于虫卵抗原的研究,提示虫源性分子可通过抗原特异性的模式识别受体(pattern recognition receptors, PRRs)级联通路的靶向作用,启动并支配获得性免疫应答的发展。由此不难推测在血吸虫感染的早期,虫源性抗原分子也会通过相应PRRs通路引起类似的效应。然而,国内外尚缺乏全面深入地对日本血吸虫感染后的天然免疫应答特征及其相关分子机制研究的报道。鉴于Toll样受体(Toll-like receptors, TLRs)在宿主早期免疫识别中的重要作用,故在血吸虫感染的天然免疫应答研究中应予特别关注。在对曼氏血吸虫的研究中提示TLR2和TLR4是识别虫卵抗原组分的PRRs,TLR4在识别尾蚴抗原分子中也发挥重要作用。因此,本研究旨在探讨TLR2与TLR4在日本血吸虫急性感染中的作用及相关分子机理。本研究采用TLR2基因敲除(TLR2-/-)与TLR4基因发生无效突变的(C57BL/10SCNJ,即TLR4-/-)小鼠,前者以野生型C57BL/6J(B6)小鼠为对照,后者以野生型C57BL/10J(B10)小鼠为对照,建立日本血吸虫急性感染的小鼠模型。为了观察感染/致病结果,在感染后6w通过胸主动脉灌注法计数成虫数、消化肝脏计数虫卵数以及每对成虫产卵数;为了观察感染后宿主的整体免疫应答特征,以间接ELISA法检测血清中日本血吸虫特异性IgG抗体,以Bio-plex技术检测脾脏单个核细胞培养上清中Th1/Th2型细胞因子的表达水平;进而采用Affymetrix公司的小鼠基因组430 2.0基因芯片,全面比较分析感染后6w小鼠脾脏单个核细胞内表达基因的转录水平,对信号强度发生2倍及以上变化的差异基因进行GO及pathway的显著性分析,并以RTQ-PCR法加以验证;最后采用虫源性抗原SEA致敏小鼠,观察比较小鼠脾脏自然杀伤细胞(Natural Killer cells, NK细胞)与CD8+T细胞的免疫杀伤功能。本研究获得如下主要结果:1、在日本血吸虫急性感染过程中,与野生型小鼠相比,TLR2-/-小鼠虫荷与卵荷均降低,而TLR4-/-小鼠的虫荷与卵荷则增高。在日本血吸虫感染后6w,TLR2-/-组的成虫数、肝脏总虫卵数以及每对成虫产卵数均低于B6组;而与B10组相比,感染后TLR4-/-组小鼠的成虫数、肝脏总虫卵数以及每对成虫产卵数均高于B10组。其中,仅成虫数未显示统计学差异,而肝脏总虫卵数以及每对成虫产卵数均具有显著性差异。2、在日本血吸虫感染6w后,TLR2-/-小鼠的细胞免疫应答水平较B6小鼠显著增强,而TLR4-/-小鼠的细胞应答水平低于B10小鼠。脾脏单个核细胞经虫源性抗原SEA或有丝分裂原ConA刺激后,培养上清中细胞因子水平显示:在SEA和ConA刺激下,TLR2-/-小鼠产生IL-12p70、IFN-γ、IL-2、GM-CSF和IL-4的水平均较B6小鼠显著增高;在ConA刺激下TNF-α和IL-10较高,而在SEA刺激下产生的IL-10低于B6小鼠。在SEA与ConA刺激后,TLR4-/-小鼠产生IL-12p70、IFN-γ、IL-4和IL-5的水平均显著低于B10小鼠;在单独ConA刺激下IL-10显著降低,而在SEA刺激后,TNF-α显著降低。其余细胞因子在TLR4-/-亦低于B10小鼠,但无统计学上差异。3、日本血吸虫感染后,TLR4-/-小鼠抗原特异性IgG水平显著高于B10小鼠,但TLR2-/-小鼠与B6小鼠之间无显著差异。随着日本血吸虫感染的发展,小鼠血清中日本血吸虫特异性IgG抗体水平均持续升高。在感染后3w和6w,TLR4-/-小鼠血清中针对可溶性成虫抗原(SWAP)和SEA特异的IgG抗体水平显著高于B10组;但TLR2-/-与B6小鼠之间无显著性差异。4、采用高通量基因芯片技术分析比较日本血吸虫感染6w后脾脏单个核细胞的基因转录水平,发现TLR2-/-小鼠中与T细胞免疫应答相关的大量基因以及免疫杀伤功能相关的基因(颗粒酶、穿孔素、FasL等)被显著上调;而在TLR4-/-小鼠中,这两大类基因的表达均被显著下调。与B6小鼠相比,在感染后6w的TLR2-/-小鼠脾细胞中,有251个基因信号有显著性改变,并且信号强度增强2倍以上,444个基因信号减弱2倍以上;在TLR4-/-小鼠脾细胞中,仅有91个基因信号增强2倍以上,同时121个基因信号减弱2倍以上。值得注意的是,在TLR2-/-小鼠中信号增强的基因在TLR4-/-小鼠中大多减弱。对差异基因的GO分类及pathway的显著性分析发现,差异基因及参与的通路主要涉及两大类:一是T细胞免疫应答相关基因及通路,二是免疫杀伤相关基因及通路。5、经SEA致敏后,TLR2-/-和TLR4-/-小鼠脾脏NK细胞杀伤活性均略高于野生型小鼠,但无显著性差异;相较于野生型小鼠,CD8+T细胞杀伤功能相关基因mRNA水平在TLR2-/-小鼠中升高,在TLR4-/-小鼠中降低。经SEA致敏后,TLR2-/-和TLR4-/-小鼠脾脏CD49b+NK细胞对YAC-1细胞的杀伤活性均略高于野生型小鼠,但无统计学差异。TLR2-/-小鼠CD8+T细胞中免疫杀伤功能相关基因(gzma、gzmb、gzmk、pfr1以及fasl)的mRNA水平高于B6小鼠,其中pfr1具有显著性差异;而TLR4-/-小鼠CD8+T细胞中gzma、gzmb和fasl的mRNA水平均显著低于B10小鼠。综上,感染日本血吸虫后,与野生型小鼠相比,TLR2-/-小鼠与TLR4-/-小鼠在感染结果、T细胞免疫应答程度以及免疫杀伤功能等方面均明显不同,表明TLR2与TLR4对血吸虫感染转归的影响与T细胞免疫应答以及免疫杀伤功能相关。更多还原

【Abstract】 Though great efforts have been made in the past 50 years, schistosomiasis japonica remains a serious public health problem in China. The comprehensive measures for controlling schistosomiasis were based on the population chemotherapy with praziquantel, combined with public health promotion and killing snails in endemic areas, which have made great achievements. However, the control task is being challenged by the change of natural circumstance and decreased financial resources, and the most important challenge is high rate of reinfection after chemotherapy due to large numbers of reservoir hosts and difficulties in controlling the areas with Oncomelania hupensis infestation. In 1980s, the worldwide scientists reached consensus that is the priority to develop an effective anti-schistosomiasis vaccine in order to prevent reinfection after chemotherapy.During the past decades, vaccine development and related basic immunological researches were mainly concerned on schistosomiasis mansoni. Consequently, the development of vaccines against schistosomiasis japonica is far behind that of schistosomiasis mansoni, especially the understanding of basic immunology, which greatly limits our choice of rational strategies for vaccine development for schistosomiasis japonica. Thus, fundamental immunological research should be highlighted to develop effective and specific vaccines against schistosomiasis japonica. Many vaccine studies for schistosomiasis mansoni have estabilished that antigen-specific CD4+ T cell-mediated immunity and humoral response are fundamental to the acquired resistance against schistosome, which concentrate on the acquired immunity. On the other hand, the understanding of innate immunity involved is just in the very initial stage.So far, many studies of the innate immune response during the infection of Schistosoma mansoni (S. mansoni) focused on egg antigen to illuminate why host developed a predominant Th2 response after egg deposition. All these data suggested that antigens derived from parasite could activate the cascade pathway induced by pattern recognition receptors (PRRs) to initiate and direct the acquired immune response. Thus, we could speculate that during the early stage of infection, parasite molecules might also cause a similar effect through specific PRRs pathway. Nevertheless, up to now few studies reported characteristics of immune response and related molecules during TLRs recognition of Schistosoma japonicum (S. japonicum). Those results from studies of S. mansoni also suggested that, Toll-like receptor 2 (TLR2) and TLR4 were two important immune receptors involved in recognition of components of SEA; and TLR4 also played an important role in the immunorecognition of cercaria molecules. Therefore, the purpose of this study is to investigate the effects of TLR2 and TLR4 on the outcomes of S. japonicum infection.TLR2 knock out (TLR2-/-), TLR4 null mutated (TLR4-/-) mice and their counterpart wild-type (WT) C57BL/6J and C57BL/10J mice were infected with S. japonicum. Mice were sacrificed to measure the parasite burdens at 6 weeks after infection.To evaluate the immune responses after infection, the levels of schistosoma antigen- specific IgG were determined for each mouse by indirect ELISA; and the production of Th1/Th2 cytokines by spleen mononuclear cells was determined by Bio-plex system. Then we further compared the gene expression profiles from gene deficient and their WT control 6 weeks post infection using microarrays (Mouse Genome Expression 430 2.0, Affymetrix Co.). Those genes with more than 2-fold change were further analyzed with both GO and pathway anaylsis, and the mRNA levels of cytotoxicity associated genes were validated by real-time quantitative PCR (RTQ-PCR). Finally, the cytotoxicity of natural killer (NK) cells and CD8+T cells from each groups of mice primed with SEA were compared.The main results we got are as follows:1. Both worm and egg burdens were reduced in TLR2-/- mice, but increased in TLR4-/- mice. Six weeks after challenge with 40 S. japonicum cercariae, parasite burdens including total worms, total eggs in livers and eggs produced by each pair of worms (EPP) were evaluated. The parasite burdens were decreased in TLR2-/- group, but increased in TLR4-/- group when compared to the WT mice, respectively. Significant differences were observed in total eggs and EPP but not in total worms.2. The level of cellullar immune response in TLR2-/- mice was higher than that in B6 mice, while the expression of cytokines in TLR4-/- mice was lower than that in B10 mice during the acute phase of S. japonicum infection. In the supernatant of spleen mononuclear cells stimulated with SEA or ConA, the concentrations of IL-12p70, IFN-γ, IL-2, GM-CSF and IL-4 in TLR2-/- mice were markedly higher than those of B6 mice. The production of TNF-αand IL-10 in TLR2-/- mice were more than that in WT mice when stimulated with ConA, but the expression of IL-10 stimulated with SEA was less than that in B6 mice. On the contrary, the production of IL-12p70, IFN-γ, IL-2, IL-5 and IL-4 in TLR4-/- mice were significantly less than that in B10 group; the levels of IL-10 stimulated with ConA was much less. After stimulated with SEA, the expression of TNF-αwas significantly less than that in the B10 mice.3. After infection with S. japonicum, the level of antigen-specific IgG in TLR4-/- mice was significantly higher than that in B10 mice, but there was no significant difference between TLR2-/- and B6 mice. Following S. japonicum infection, schistosome antigen-specific IgG antibody in the sera of all three groups of mice kept on rising. In acute phase of infection, the level of SWAP- and SEA-specific IgG antibody in TLR4-/- mice were significantly higher than those in B10 mice; but there were no significant difference in both SWAP and SEA-specific antibody between TLR2-/- and B6 mice.4. Using the microarrays to compare the gene expression profiles in splenocytes 6 weeks post-exposure, we found that two categories of genes involved in T cell responses and cytotoxicity were significantly upregulated in TLR2-/-mice; and these genes were downregulated in TLR4-/- mice. In TLR2-/- mice, there were 251 probe sets found to have significantly higher signal values and 444 probe sets were downregulated when compared to B6 mice; and in TLR4-/- mice, only 91 probe sets were found to be enhanced more than 2-fold, and 121 genes have lower signal values. Interestingly, those genes enhanced in TLR2-/- mice were downregulated in TLR4-/- mice correspondingly. Further analysis of GO and pathway indicated that these genes and pathway mainly belong to two categories: T cell response and cytotoxicity pathway.5. After being primed with SEA, the cytotoxicity of NK cells in both TLR2-/- and TLR4-/-mice was slightly higher than that in WT mice without significant difference. And the mRNA levels of cytotoxicity-associated genes from CD8+T cell were increased in TLR2-/- mice, but decreased in TLR4-/- mice. The cytotoxicity of CD49b+ NK cells from spleens of both TLR2-/-and TLR4-/-mice to YAC-1 cells was similar to that in their WT controls. On the other hand, the mRNA levels of cytotoxicity-associated genes (gzma, gzmb, gzmk, pfr1 and fasl) from CD8+ T cells of TLR2-/- mice were higher than that of B6 mice, and only the level of pfr1 was significantly different; and the expression of gzma, gzmb and fasl from CD8+ T cells of TLR4-/- mice were significantly lower than those of B10 mice.In conclusion, after infection with S. japonicum, parasite burdens, T cell-mediated immune response as well as the cytotoxicity pathway were significantly different in TLR2-/- mice and TLR4-/- mice, when compared with the wild-type mice, respectively. These results indicate that TLR2 and TLR4 might influence the outcomes of S. japonicum infection through both T cell immune response and cytotoxicity pathway.更多还原