他克林体外抗细粒棘球蚴作用研究

目的 研究乙酰胆碱酯酶抑制剂他克林体外抗细粒棘球蚴的作用效果。方法 体外分离和培养细粒棘球蚴原头节和生发层细胞,经浓度为4、8、10、20和40 μg/mL的他克林作用3 d后经0.1%亚甲基蓝染色观察,记录死活原头节的数目以计算原头节死亡率,另外用CCK-8试剂盒检测药物对生发层细胞活性的影响并计算细胞活性抑制率。同时用透射电镜和扫描电镜分别观察药物对细粒棘球蚴原头节和生发层细胞超微结构造成的影响。结果 经20 μg/mL和40 μg/mL的他克林体外作用3 d后,原头节的死亡率高达100%,其中死亡原头节的体壁出现轻微肿胀且伴随着外部轮廓的消失,同时超微结构亦发生明显改变,原头节体壁组织中出现了大量的空泡和脂肪滴。当他克林浓度为40 μg/mL时,可造成生发层细胞全部死亡。细粒棘球蚴生发层细胞粘附于培养介质的基质消失、细胞发生聚集且数目减少。扫描电镜可观察到他克林作用3 d后的细胞出现塌陷或者萎缩。结论 他克林可直接影响体外培养的细粒棘球蚴原头节和生发层细胞的活性,是潜在的包虫病治疗药物。

Efficacy of tacrine on Echinococcus granulosus in vitro

In order to study the effects of tacrine as the acetylcholinesterase inhibitor on Echinococcus granulosus in vitro, the E. granulosus protosoleces and germinal cells were isolated and cultured in vitro. The protoscoleces and germinal cells were incubated with tacrine at the concentration of 4, 8, 10, 20 and 40 μg/mL over 3 days respectively. Then the protoscoleces were stained with 0.1% methylene blue and the percentage of vital protoscoleces were calculated from the number of live and dead parasites. And the inhibition rate of germinal cells were determined based on CCK-8 kit. At the same time, the ultrastructure changes of parasites were observed through transmission and scanning electron microscopy. The results showed that after 3 days of exposure to 20 and 40 μg/mL of tacrine, the percentage of dead protoscoleces was 100%, and the slight swelling of the soma region and the outline of tegument dispersed became evident in the dead protoscoleces. In addition, the ultrastructural alterations was obvious including the presence of numerous vacuoles and lipid droplets in the tegument. The activity inhibition rate on germinal cells was about 100% at 40 μg/mL tacrine, the disappearance of adhesion matrix, the aggregation and detaching from the culture plate of E. granulosus germinal cells had be seen as well. SEM revealed the contraction and collapse of germinal cells after tacrine treatment. It suggests that tacrine could directly effect the activity of E. granulosus protocoleces and germinal cells in vitro. Therefore, tacrine is a potential drug in the treatment of hydatid diseases.