摘要
本文旨在探究体外培养日本血吸虫幼虫的新方法、吡喹酮透皮给药药效分析,以及感染日本血吸虫和吡喹酮治疗后宿主的免疫应答反应。不仅为日本血吸虫幼虫的体外操作提供了新方法,也为治疗血吸虫病提供更为便利的治疗途径,同时还探讨了吡喹酮与炎症反应的关系,为吡喹酮作用机理的研究提供了部分理论依据。
无菌摘取感染日本血吸虫尾蚴42d的BALB/c小鼠肝脏若干,组织均质仪匀浆,恒温孵化毛蚴,将毛蚴与草地夜蛾(Spodoptera frugiperda)卵巢细胞(sf9细胞)于恒温细胞培养箱中共培养。分别拍摄共培养1h、12h、24h及3d毛蚴生长照片。将培养3d的母胞蚴注射于阴性钉螺头足部,80d后观察钉螺逸蚴情况。本试验重复三次,三次试验的钉螺存活率分别为24.72%、28.23 %和57.89%,活螺阳性率分别达4.55%、8.57%和14.29%。上述试验逸出的尾蚴经人工感染BALB/c小鼠,检测尾蚴的活力,同时通过组织化学染色法和PCR方法鉴定尾蚴的相关基因特性。
BALB/c小鼠经腹部贴片人工感染尾蚴40±2条,感染35d后,按800mg/kg、480mg/kg、240mg/kg经吡喹酮透皮剂各治疗一次,以及240mg/kg透皮3次,腹部透皮治疗小鼠,一周后解剖小鼠,统计减虫率、减肝卵率和减肝脏虫卵孵化率等指标。最高减虫率到达50%以上,800mg/kg透皮剂量的减肝卵率接近70%,各组减雌率均为100%,且减毛蚴孵化率均在50%以上。
同时,以BALB/c小鼠作为实验动物模型,BALB/c小鼠经腹部贴片人工感染尾蚴30±2条。在感染28d和35d使用吡喹酮口服制剂对其进行定量(900mg/kg)治疗,通过流式细胞术检测各试验组小鼠CD4~+、CD8~+淋巴细胞亚群比例,以及IL-4、IFN-γ、IL-17等细胞因子表达水平的变化。
试验结果表明,经本试验培养阳性钉螺释放的尾蚴具有感染性,且与日本血吸虫有相同的基因特性。以sf9细胞作为滋养层细胞体外培养毛蚴,通过显微注射获得人工培养阳性钉螺,为日本血吸虫幼虫的体外操作提供了一种新的方法。吡喹酮透皮剂杀灭血吸虫的效果具有剂量和次数依赖性,少量多次的治疗效果较好,各组均达到分离雌雄虫合抱的效果。吡喹酮治疗后,因虫体的死亡引发了一系列炎症反应,诱发了Th1/Th2型混合的细胞免疫应答,其中以Th2型免疫反应为主。Th17细胞在炎症反应中也发挥了重要的作用,吡喹酮对炎症反应可能有抑制作用。
This paper aimed to explore a new method of in vitro cultivation of Schistosoma japonicum larvae,transdermal delivery efficacy of praziquantel,the immune response of the host of Schistosoma japonicum and after treatment with praziquantel. Not only provided a new method of in vitro manipulation of Schistosoma japonicum larvae, but also provided a more convenience therapeutic approach for the treatment of schistosomiasis, explored the relationship between praziquantel and inflammation, in order to provide some theoretical basis for studying the mechanism of praziquantel.
Several BALB / c mouse`s livers were removed aseptic which were infected by Schistosoma japonicum after 42 days, homogenated by tissue homogenate instrument, incubated miracidium with constant temperature, the miracidium co-cultivated with Spodoptera frugiperda ovary cells (sf9 cells) in constant temperature Cell incubator. The pictures of miracidia were recorded after co-cultivation for 1 h, 12 h, 24 h and 3 d. The co-cultivated for 3 days mother sporocysts injected in the head and foot of negative snails, after 80 days, observed whether cercariae could be obtained or not. The experiment repeated three times, the survival rates of snails in three experiments were 24.72%, 28.23 % and 57.89% respectively while the positive rates of cercariae were 4.55%, 8.57% and 14.29% respectively. The related gene characteristics of cercariae were identified by histochemical examination and PCR method.
BALB / c mice were infected with Schistosoma japonicum cercariae 40±2.After 35 days, mice were treated with different concentration of PZQ transdermal agent, 800mg/kg, 480mg/kg, 240mg/kg individually, and treated with different times of PZQ transdermal agent, 240mg / kg once and three times individually.percutaneous treatment of abdominal After one week, mice were dissected, statistical of worm reduction rate, egg reduction rate and hatch of egg reduction rate and so on. By treating with 800mg/kg of PZQ transdermal agent, worm reduction rate up to 50%, egg reduction rate up to 70%, and each group of female reduction rate was 100%, miracidia reduction rate up to 50%.
Meanwhile, BALB/c mice were experimental animal models. BALB / c mice were infected with cercariae 30±2. After 28 days and 35 days of infection, some of mice were treated with praziquantel with quantitative (900mg/kg) by oral approach. After one week or two weeks of treatment, the ratio of CD4+, CD8+ lymphocyte subsets, and the expression levels of cytokines IL-4, IFN -γ, IL-17 were detected by flow cytometry in mice.
Animal experiments showed that the cercariae released from artificial cultivation of positive snails were infectiuous and had the same gene characteristics of Schistosoma japonicum. Sf9 cells could be feeder cells for in vitro-coltivation of miracidia and positive snails could be obtained by in vitro micro-injection of cercariae thus to provide a new approach for in vitro operation of Schistosoma japonicum larvae. The effect on Schistosoma was depended on dose and times of praziquantel transdermal agent, the better treatment effect was more times and low dose, each group reached the effective of separating the male and female worms. After praziquantel treatment, a series of inflammatory reactions were caused by the death of worm, induced a mixed immune response with Th1/Th2 type, which mainly was Th2 type immune response. Th17 cells also played an important role in the inflammatory response. Praziquantel could inhibit the inflammatory response.
引文
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