【作者】 王婷；

【导师】 李雍龙；

【作者基本信息】 华中科技大学 ， 病原生物学， 2013， 博士

【摘要】 血吸虫病是一种严重危害人类健康的疾病,而血吸虫尾蚴发现宿主并主动钻入宿主体内是寄生虫与宿主长期共进化过程中产生的一种生物学适应性行为,对此行为的深入了解不仅有助于进一步认识寄生虫与宿主的相互关系,而且可为血吸虫病的预防、早期诊断及抗血吸虫感染的疫苗研究提供新的靶点。本课题重点研究了血吸虫尾蚴入侵宿主过程中的“延迟脱尾”现象及其临床意义,研究内容分为如下4个部分：一、日本血吸虫尾蚴“延迟脱尾”现象的研究传统观念认为,血吸虫的尾蚴在入侵宿主皮肤过程中会脱去尾部。虽然很早就有学者利用组织切片证明曼氏血吸虫尾蚴在钻穿小鼠皮肤时会带着尾部一起进入,但由于实验中使用的尾蚴数量过大,超过了自然感染的可能数量,故此结果一直未被学界接受。近来,Whitfield等人在应用Franz cell装置体外研究曼氏血吸虫尾蚴钻穿人类皮肤行为时发现,尾蚴在侵入宿主皮肤时尾部并未完全脱落,有相当一部分尾蚴的尾部随同体部一起进入了宿主皮肤组织,他们把这一现象称为尾蚴的“延迟脱尾”(delayed tail loss, DTL)[3,4,5]。该研究表明,曼氏血吸虫尾蚴钻穿宿主皮肤时尾部随同体部一起进入皮肤是一种自然行为,即使在自然感染量的情况下仍能发生。由于不同种属血吸虫行为学差异很大[6],因此不能由此推论其他血吸虫尾蚴在钻皮时也具有“延迟脱尾”现象。实际上,毛毕吸虫(Trichobilharzia szidati)就不具有“延迟脱尾”现象,Haas和Haeberlein发现毛毕吸虫在钻穿宿主皮肤前大约0—105秒就会脱去尾部。日本血吸虫尾蚴在钻穿宿主皮肤时是否具有“延迟脱尾”现象尚未见报道,为此,我们利用Franz cekk[4]装置观察了日本血吸虫尾蚴钻穿小鼠皮肤的情况,并进一步研究了“延迟脱尾”现象与不同尾蚴感染量之间的关系。研究结果显示,日本血吸虫尾蚴在钻穿小鼠皮肤时也存在“延迟脱尾”现象,而且证实日本血吸虫在钻穿小鼠皮肤时,其“延迟脱尾”的比率在一定范围内与感染尾蚴密度有关,即尾蚴密度从10条/2.25cm2到100条/2.25cm2范围内,延迟脱尾率随着感染尾蚴密度升高而上升,而当尾蚴密度大于100条/2.25cm2时,延迟脱尾率达峰值且不再随感染密度升高而上升,即日本血吸虫的“延迟脱尾”现象在一定范围内具有密度依赖性。在上述研究的基础上,本课题继续探讨了日本血吸虫“延迟脱尾”这一生物学现象与临床的关系。二、日本血吸虫“延迟脱尾”与尾蚴性皮炎的关系研究血吸虫病是一种免疫性疾病,在血吸虫感染过程中,尾蚴、童虫、成虫和虫卵均可对宿主造成损害,损害的主要原因是血吸虫不同虫期释放的抗原均能诱发宿主的免疫应答,从而造成一系列的免疫病理变化,其中尾蚴所致的损害主要表现为入侵的局部皮肤出现丘疹,局部毛细血管扩张充血,伴有出血、水肿和嗜酸性细胞浸润。尾蚴性皮炎的发生和严重程度常与感染的次数有关,初次感染者皮肤反应通常不明显,重复感染者反应逐渐加重,严重者可出现全身水肿和多形红斑[9]。Kourilova等利用禽类的血吸虫—毛毕吸虫(Trichobilharzia regenti)感染哺乳动物,证实在皮内死亡的童虫是引起尾蚴性皮炎的主要原因,Kourilova和Lichtenbergova[10,11]也发现,在尾蚴性皮炎病人的皮肤内,入侵的虫子很快就死亡了,提示尾蚴性皮炎的发生与寄生虫和宿主的相容性有关,当血吸虫尾蚴入侵非正常宿主时更易引起尾蚴性皮炎,事实也证明在我国引起尾蚴性皮炎的主要是寄生于禽类的多种毛毕吸虫(Trichobilharzia)和寄生于牛的东毕吸虫(Orientobilharzia)[12]。人类和多种哺乳动物是日本血吸虫是的适宜宿主,尾蚴钻穿皮肤后转化为童虫,大部分童虫可经血管或淋巴管进入血循环进一步发育,仅部分童虫在由淡水环境进入恒温的血清—组织液环境时,会受到损伤而死亡。3种常见的人类血吸虫(日本血吸虫、曼氏血吸虫和埃及血吸虫)尾蚴在初次钻穿宿主皮肤后,童虫的死亡率以日本血吸虫的最低(7%),其次是曼氏血吸虫(28-43%)和埃及血吸虫(30%)。鉴于我们先前的研究发现日本血吸虫尾蚴钻穿宿主皮肤时具有“延迟脱尾”现象,故本研究拟探讨与人类和多种哺乳动物高度相容的日本血吸虫引起的尾蚴性皮炎是否与滞留在皮内的尾部有关。本研究利用日本血吸虫尾蚴、尾蚴体部和尾部分别刺激已感染过同种血吸虫的小鼠,观察尾蚴身体的不同部分与尾蚴性皮炎发生的关系以及相关细胞因子的变化。结果显示,经日本血吸虫尾蚴和尾蚴尾部刺激的小鼠,其皮肤的炎性反应最重,而经尾蚴体部刺激的小鼠皮肤与生理盐水对照组无显著型差异。经日本血吸虫尾蚴和尾蚴尾部刺激的小鼠主要表现为局部皮肤呈不同程度的红色丘疹,组织切片显示局部水肿和嗜酸性粒细胞增多。血清细胞因子检测显示经日本血吸虫尾蚴和尾蚴尾部刺激小鼠的IFN-γ,IL-4和IL-5的含量明显增高。本研究结果提示,日本血吸虫尾蚴引起的皮肤炎性反应主要与滞留在皮内的尾部有关。由于Th2型细胞因子(IL-4和IL-5)与炎症反应有关,而IFN-γ是迟发性过敏反应的重要介质,因此,在皮内死亡、崩解的尾部引起的皮炎可能是过敏反应和炎症反应的结果。三、日本血吸虫尾蚴“延迟脱尾”与早期诊断的关系研究血吸虫病是一种严重危害人类健康的寄生虫病,其危害性主要是由虫卵引起的,沉积在肝脏中的虫卵是引起肝纤维化及其后续病变的主要原因。粪检发现虫卵是目前判断血吸虫感染的唯一可靠方法,但粪检发现虫卵也意味肝脏的损害即已开始且已具有传播潜能,因此不具早期诊断的价值。日本血吸虫通常在感染后24天开始产卵,由于吡喹酮能有效杀死成虫,因此如能在成虫产卵前作出正确诊断,适时足量用药,则不仅能达到控制血吸虫病发生、发展的目的,且能阻断血吸虫病的传播。在血吸虫病诊断中,血清学方法较病原学方法更敏感且能更早地检测到相应的抗体。但目前国内外常用的诊断抗原鲜能在感染早期(感染后2周)检测出相应的抗体,因此早期诊断成为血吸虫病防治中的“瓶颈”问题,寻找一种能符合早期诊断要求的抗原是解决血吸虫病防治的关键。在“日本血吸虫尾蚴延迟脱尾”研究中,我们发现尾蚴在钻穿宿主皮肤时大部分尾蚴并未脱去尾部,而是整体进入宿主皮内,然后将尾部滞留在皮内,而童虫循皮下的血管和淋巴管移行至肺部和肝脏。由于尾部在皮内滞留继而崩解,因此我们推测首先被抗原提呈细胞提呈和处理的应是在皮内停留并崩解的尾部释放的蛋白组分,如果如此,则宿主最早出现的应是针对尾蚴尾部抗原的特异性抗体。为此,本研究探讨了尾蚴尾部抗原在血吸虫病早期诊断中的价值。本研究按常规方法收集尾蚴并分离尾部和体部,制备全尾蚴抗原(SWA)、尾蚴体部抗原(SBA)和尾蚴尾部抗原(STA),用常规ELISA法检测感染后不同时间小鼠血清相应抗体。结果显示,感染后2周,STA检测组的血清0D值显著高于其他组,且阳性检出率达100%(10/10),而SCA组合SBA组分别于感染后3周和4周期阳性检出率才达100%(10/10)。宿主对血吸虫特异性反应的启动有赖于对抗原提呈细胞的募集以及抗原提呈细胞对血吸虫抗原的处理和呈递,此过程在尾蚴进入皮肤后即可发生。Sato&Kamiya证实,即使尾部仅进入表皮层,表皮的未成熟树突状细胞(Langerhans cells)也能发挥抗原提呈作用并刺激皮肤引流淋巴结的T细胞,进而诱导相应的免疫应答。在本研究中我们也发现最早能被检出的抗体是针对尾蚴尾部粗抗原的抗体,提示感染日本血吸虫的宿主体内最早出现的特异性免疫反应是由滞留在皮内的尾部引起的,因此日本血吸虫尾蚴的尾部抗原较全尾蚴抗原更具早期诊断的价值。如能利用蛋白重组技术人工合成尾部有效的抗原组分,则能为解决血吸虫病的早期诊断问题。四、日本血吸虫断尾尾蚴的感染性研究血吸虫尾蚴由体部和尾部组成,体部具有2对腺体,即头腺和钻腺,尾部分尾干和尾叉。在水中自由生活的日本血吸虫尾蚴,静止时以腹部朝上、尾部向背部弯曲漂浮在水面,活动时依靠尾部的摆动而作以尾部向前的倒退姿势运动。尾蚴识别宿主是血吸虫感染的关键,血吸虫尾蚴发现宿主是通过识别宿主所发出的信号。尾蚴具向光性,因此常常集中在水面,然后通过对宿主发出的热梯度(thermal gradient)或化学信号的感应而后依靠尾部的摆动趋向宿主。血吸虫尾蚴钻穿宿主皮肤是血吸虫感染的关键。传统观点认为,血吸虫钻穿宿主皮肤时是依靠腺体分泌物的酶促作用,体部的强烈收缩活动和尾部的摆动而进入宿主体内的,而对失去尾部的尾蚴是否仍然具有感染性尚未见报道。本研究利用体外机械断尾后的尾蚴体部按常规方法经腹部皮肤感染小鼠,感染后45天经左心室灌注冲虫,收集虫体观察其发育及生殖状况并摘取小鼠肝脏观察病理变化。结果发现,断尾尾蚴仍具有感染性。正常尾蚴成功感染的平均机率为47.5%,而断尾尾蚴成功感染的平均机率为10%,且断尾尾蚴入侵宿主后仍能沿正常途径生长、发育和繁殖。正常尾蚴发育成熟的雌、雄成虫其平均长度分别为14.2±2.47和10.5±2.26mm,而断尾尾蚴发育成熟的雌雄成虫分别为12.8±0.73和9.15±0.85mm,两者无显著性差异(>0.05)。断尾尾蚴发育成熟的雌虫其产卵量与正常尾蚴发育成熟的雌虫的产卵量分别为6947.5±2155.5和6245.8±2012.9,两者也无显著性差异(p>0.05)。肝脏病理学观察,发现两者均可引起典型的血吸虫性肝脏病变,主要表现为镜下可见多个局限、境界清楚的肉芽肿,其内可见较多的嗜酸性粒细胞,纤维母细胞及少许淋巴细胞。本研究结果提示,尾部有助于尾蚴的钻穿行为,但失去尾部的尾蚴仍然具有感染性且可导致血吸虫性肝脏病变,因此在疫水调查或在研制以尾蚴为靶标的杀虫剂时,对失去尾部的尾蚴的感染作用应慎重评价。更多还原

【Abstract】 Schistosomiasis is a serious disease to human. The parasite finding and invading human are adaptive behaviors developed over a long period of evolution. Understanding well the behaviors is not only helpful to the relationship between the parasite and host but also favor to control of the parasitic disease. In the project, we focus on studies on delayed tail loss of Schistosoma japonicum and its clinical significance.The research contents included the following4parts:Prat1. Studies on delayed tail loss of Schistosoma japonicum cercariaAim:Traditionally, schistosome cercariae would shed their propulsive tails as they penetrate the skin of host. While recently, contrary to this long-held assumptions, some new researches have shown that Schistosoma mansoni do not shed their tails at the onset of penetration. This phenomenon is termed as delayed tail loss (DTL). In the present study, we intend to explore DTL of Schistosoma japonicum cercariae.Methods:using a device Franz cell to observe the behavior of Schistosoma japonicum cercariae while S. japonicum cercariae penetrated a mouse skin in vitro.Rresults:DTL also happens with S. japonicum cercariae during penetration of mouse skin. It occurred at all cercarial densities tested from as few as10cercariae/2-25cm2of mouse skin up to200cercariae. Furthermore, it was demonstrated that there was a density-dependent increase in DTL as cercarial densities increased.Conclusion:The phenomenon of DTL also occurs in S. japonicum cercariaePart2. Studies on the relationship between DTL of S. japonicum cercariae and cercarial dermatitisAim:It has been demonstrated that cercarial dermatitis was caused by schistomulum died in skin. This research is to explore the relationship between cercarial dermatitis and tails lost in skin.Methods:The mice infected Schistosoma japonicum were injected with cercariae, cercarial bodies and tails respectively. The relationship between the dermatitis and cercaria, cercarial body and cercarial tail were observed, meanwhile, the cytokines IFN-γ, IL-4and IL-5were detected.Results:The local skin of mice stimulated by tails showed the red papules, tissue edema and eosinophilie infiltration, the levels of IFN-γ, IL-4and IL-5increased compared with the mice stimulated by cercariae or cercarial bodies.Conclusion:The tails lost in the host skin is involved in S. japonicum cercarial dermatitis, the inflammatory reaction in local skin is related with IL-4and IL-5, and the anaphylactic reaction is related with IFN-y. Part3. Studies on the effect of DTL of S. japonicum cercariae on detection of the early infectionAim:Schistosomiasis is a serious disease to human being, the major harm to human is induced by eggs. The laying eggs deposited in the liver cause fibrosis of the liver. Since Praziquntal can kill the adult worms, it is possible to eradicate the infection if the infection can be detected, if it can be done before laying eggs, the infected people can be protected from the damage of eggs. In this case, early diagnosis is very important for schistomiasis control. The aim of the research focuses on to identify an antigen which can early detect the corresponding antibody.Methods:Preparation of crude antigens from whole cercariae (SWA), the bodies of cercariae (SB A) and tails of cercariae (STA) respectively. Detection of the antibodies from the sera of mice infected with schisotome and evaluation of the value of the antigens to identify the early infection.Results:The levels of antibodies against STA is much higher than those against SWA or SB A (P<0.05), and100%of positive reaction is available2weeks post infection using STA detection.Conclusion:STA is superior to detect the early infection comparied with SWA and SBA.Part4. Studies on the infectious ability of Scistosoma japonicum cercariae without their tails.Aim:To explore the infectious ability of Scistosoma japonicum cercariae which lost their tails. Methods:Collect the cercariae lost their tails and infect the mice through the abdominal skin as routine way. Collect the adult worms and mice livers45day after infection. Comparison of the development and reproduction of the worms in the mice infected with normal cercariae and cercariae without tails. The pathological changes of the mouse liver after infected with cercariae without tails were performed.Results:The cercariae lost their tails still possess infectivity even though the successful infection is lower than that of the normal cercariae (10%vs47.5%). It has been demonstrated that the cercariae lost tails could develop normally and cause a typical schistosome pathological changes in mice livers. Conclusion:The cercariae lost their tails still possess infectivity, therefore it should be careful to evaluate their effects on investigation of infest water or cercaricide.In conclusion, the innovations of the research are:1. The phenomenon of DTL occurs in S. japonicum cercariae and it was demonstrated that there was a density-dependent increase in DTL as cercarial densities increased.2. The cercarial tails lost in the host skin is involved in S. japonicum cercarial dermatitis.3. Crude antigens from tails of cercariae (STA) are superior to detect the early infection compared with those from whole cercariae (SWA) and bodies of cercariae (SBA).4. The cercariae lost their tails still possess infectivity.更多还原