关键词: 细粒棘球绦虫, 原头节, 过氧化氢, 细胞凋亡, 半胱天冬氨酸蛋白酶, FAS基因, 基因表达

Abstract: Objective To explore the apoptosis induced by hydrogen peroxide( H₂O₂) in protoscolex of Echinococcus granulosus. Methods Protoscoleces were cultured in vitro，and used for the experiment in 2 groups：RPMI 1640 medium and RPMI 1640 medium added with glutamine. They were then treated with different concentrations of H₂O₂ to induce apoptosis. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay (TUNEL) was employed to observe the apoptosis. Protein expression of caspase-1, caspase-3 and Fas was detected by SP immunohistochemical technique，stained with DAB re-stained with hematoxylin. A yellow or brown color nucleus revealed positive apoptosis cells in protoscolex，a brown reaction product in cytoplasm showed positive cells of caspase-1 and caspase-3，and brown cell membrane and cytoplasm revealed Fas product；otherwise it was judged as negative. According to the percentage of positive cells in a protoscolex，the expression level was divided as 4 grades. The percentage of less than 5% was regarded as “-”，5%-25% as “+”，26%-50% as “++”，more than 50% as “+++”. The experiments were repeated 2 times with controls. Results In RPMI 1640 group，positive TUNEL was found in the protoscolex induced by 1 mmol/L H₂O₂ inducing for 12 hours. Induced by 1 mmol/L H₂O₂ for 4 h，the “+ - ++”expression rate of caspase-1 and caspase-3 in the protoscoleces was 86.6% and 77.8%，and for 8 h，86.6% and 80.0% respectively，a significant increase in comparison to the control（P＜0.05). Induced by 5 mmol/L H₂O₂ for 4 hours，the “++ - +++”expression rate of caspase-1 was 93.0%，and the “+ - ++”expression rate of caspase-3 was 89.5%；induced for 8 h，the “++ - +++”expression rate of caspase-1 decreased to 53.2%，and the “+ - ++”expression rate of caspase-3 decreased to 48.4% and “-”expression rate increased to 46.8%. Under 5 mmol/L H₂O₂ for 4 h the expression rate of caspase-3 significantly decreased at 8 h（P＜0.01). In the group of RPMI 1640 plus glutamine，induced by 5 mmol/L H₂O₂ for 8 h，the “++ - +++”expression rate of caspase-3 in protoscolex was100%(P＜0.01). However，in RPMI 1640 group，induced by 5 mmol/L H₂O₂ for 8 h，the “++ - +++”expression rate of caspase-3 in protoscolex was 32.2% and 46.8% were negative. The Fas product with positive reaction in protoscolex was found in both control and induced groups：in RPMI 1640 group，under 5 mmol/L H₂O₂ induced for 4 h，“+ - ++” expression rate was 53.0% and control was 20.0%；and in the group of RPMI 1640 plus glutamine，under 5 mmol/L H₂O₂ induced for 8 h，“+ - ++” expression rate was 88.7% and control was 71.4%，increased in both groups after inducion (P＜0.05). Conclusion Apoptosis in the protoscolex of E. granulosus may be induced by H₂O₂.

Key words: Echinococcus granulosus, Protoscolex, H₂O₂, Apoptosis, Caspase, Fas ene, Gene expression