丙烯硫脲对日本血吸虫酚酶抑制作用的组织化学研究

Histochemical studies of allyl thiourea's inhibiting effect on the phenol oxidase activities in Schistosoma japonicum

目的 通过组织化学方法研究丙烯硫脲对小鼠体内、体外日本血吸虫成虫酚酶 (phenol oxidase,PO)活性的抑制作用。方法 将 4 2 d日本血吸虫活成虫置于含 2 5 mmol/L 邻苯二酚的磷酸盐缓冲液 (p H6 .8)中 ,2 8℃下孵育 30 min,取出虫体置载玻片上 ,加 2滴含 0 .1%戊巴比妥钠的 PBS溶液 ,置 L eica荧光显微镜下 ,分别在普通光照和紫外光激发下观察酚酶在虫体内的组织学定位。体外抑制实验时 ,加底物前先将成虫置于含 5mm ol/L 丙烯硫脲的 PBS溶液中 ,2 8℃下孵育 10 min;体内抑制实验时 ,在小鼠感染日本血吸虫尾蚴 2 0 d后 ,按30 0 m g/kg隔日 1次腹腔注射丙烯硫脲。结果 体外抑制实验组雌虫卵黄腺及子宫内虫卵卵壳和雄虫体壁表层的桔黄色荧光或赭褐色颜色反应均基本消失 ;体内抑制实验组雌虫子宫内未见虫卵或虫卵样物质 ,仅见大量泥沙样卵黄颗粒 ,其荧光或颜色反应也基本消失。结论 通过一种更灵敏的显示酚酶活性的组织学定位方法 ,观察到不仅雌虫含有酚酶 ,雄虫也有酚酶活性。证明丙烯硫脲对血吸虫成虫的酚酶活性有明显的抑制作用 。

Objective To observe the inhibiting effect of allyl thiourea on phenol oxidase(PO) activities in Schistosoma japonicum in vivo and in vitro by histochemical methods.Methods Forty two days fresh female and male worms were incubated for 30 min at 28℃ in pH 6.8 phosphate buffer containing substrates of 25 mmol/L catechol and 0.1% sodium phenobarbital. The distribution of PO was observed by the fluorescence microscope. At the same time, in order to observe the effect of allyl thiourea as a inhibitor of phenol oxidase of Schistosoma japonicum in vitro and in vivo, adult worms were treated by 5 mmol/L allyl thiourea for 10 min prior to catechol or the mice infected with Schistosoma japonicum were injected intraperitonealy using allyl thiourea at the dose of 300 mg/kg every other day.Results The PO activities of Schistosoma japonicum could be inhibited completely by allyl thiourea in vitro and in vivo,which resulted in that the saffron fluorescence or the reddish brown colors of PO disappeared and the formation of eggs was inhibited completely in vivo.Conclusions It is a good method to observe the distribution of PO of Schistosoma japonicum by fluorescence microscope. The inhibiting effect of allyl thiourea on PO of Schistosoma japonicum has been proved by means of histochemical methods.