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目的探讨地塞米松与三磷酸腺苷(ATP)联用在体外诱导细粒棘球绦虫原头节细胞凋亡的作用。方法体外培养细粒棘球绦虫原头节,分别设地塞米松(5mmol/L)组、ATP(1.6mmol/L)组、地塞米松(5mmol/L)+ATP(1.6mmol/L)组和空白对照组,显微镜下观察原头节变化。药物诱导8h后,选取原头节形态改变最明显的一组和空白对照组,透射电镜观察这两组原头节的超微结构,原位末端脱氧核糖核苷酸转移酶标记技术(TUNEL法)检测原头节中的凋亡细胞,半胱氨酸天冬氨酸蛋白酶-3(caspase-3)活性检测试剂盒检测该酶活性,琼脂糖凝胶电泳检测两组原头节的DNA片段。结果药物诱导8h后观察,与对照组相比,地塞米松组和地塞米松+ATP组的原头节均出现团缩、顶突内凹和体积缩小,钙颗粒明显减少且模糊不清,未见原头节活动,其中地塞米松+ATP组原头节的形态改变更明显,故选择该组作为实验组,与空白对照组进行后续试验。透射电镜观察见实验组原头节中实质细胞体积缩小、细胞膜皱缩、细胞基质浓缩、核异染色质凝集呈团块状或新月形边集于核膜下,表现出凋亡细胞的特征。TUNEL法在实验组的原头节中检测到散在的凋亡细胞,对照组则未见凋亡细胞。实验组caspase-3活性约为对照组的12倍。电泳结果显示,实验组DNA中有约150bp的核小体DNA片段。结论地塞米松与ATP联用可在体外诱导细粒棘球绦虫原头节细胞凋亡。
Objective To investigate the effect of dexamethasone and adenosine triphosphate (ATP) on inducing apoptosis of protoscoiia cells of Echinococcus granulosus in vitro. Methods The primate ceil sections of Echinococcus granulosus were cultured in vitro. Dexamethasone (5 mmol / L), ATP (1.6 mmol / L), dexamethasone (5 mmol / L) And blank control group, observe the changes of the original head under the microscope. Eight hours after the induction of the drug, the morphological changes of the primordial nodules and the blank control group were observed. The ultrastructures of these two groups were observed by transmission electron microscopy. The TUNEL method ) Was used to detect the apoptotic cells in the proroca. The caspase-3 activity assay kit was used to detect the activity of the enzyme. Agarose gel electrophoresis was used to detect the DNA fragments . Results After 8 hours of drug induction, compared with the control group, the dendrites were constricted in the proroid segments of the dexamethasone group and the dexamethasone + ATP group, and the concavities and densities of the apex were reduced. The calcium particles were obviously reduced and obscure, The activity of dendrites was not observed. The morphology of dendrites + ATP group was more obvious, so this group was selected as the experimental group and the control group was followed up. Transmission electron microscopy showed that in the experimental group, the parenchymal cells in the prothoracic nodules were reduced in size, the cell membrane was shrunk, the cell matrix was concentrated, and the heterochromatin was aggregated into clumps or crescent-shaped rim under the nuclear membrane, showing the characteristics of apoptotic cells . TUNEL method was used to detect scattered apoptotic cells in the protoscoleces of the experimental group, but no apoptotic cells in the control group. The activity of caspase-3 in the experimental group was about 12 times that of the control group. The results of electrophoresis showed that there was about 150bp nucleosome DNA fragment in the experimental group DNA. Conclusion Dexamethasone combined with ATP induces the apoptosis of protoscoleces of Echinococcus granulosus in vitro.