AZD2014对体外细粒棘球绦虫原头蚴活性影响的研究

Effect of AZD2014 on protoscoleces of Echinococcus granulosus in vitro

目的探讨双TOR抑制剂AZD2014在体外对细粒棘球绦虫原头蚴活性的影响。方法细粒棘球绦虫原头蚴分成6组进行体外培养,其中四组加入AZD2014使其浓度分别为200、100、50、25μmol/L;继续培养10 d,期间利用伊-红染色方法在倒置显微镜下观察原头蚴的活力,实验重复3次后,绘制原头蚴活力曲线;Western-blot检测100μmol/L的AZD2014培养原头蚴4 d后的p-Akt1、p-4EBP1、p-S6K1的表达量;Caspase-3试剂盒检测100μmol/L的AZD2014培养原头蚴4 d后的Caspase-3酶活性。结果 100μmol/L的AZD2014作用4 d后,原头蚴活性开始下降,存活率为(75.93%±7.28%);体外培养10 d后,100μmol/L组原头蚴活力仅为(24.03%±1.01%),200μmol/L组原头蚴活力为(2.40%±1.14%);100μmol/L的AZD2014培养4 d后,原头蚴蛋白因子p-Akt1、p-4EBP1、p-S6K1表达量明显下降;在100μmol/L的AZD2014培养原头蚴4 d后,其Caspase-3酶活性下降。结论 AZD2014在体外具有抗细粒棘球蚴原头蚴的作用,理论上,可做为临床上抗包虫的新型药物。

Objective To investigate the effect of dual TOR inhibitor AZD2014 on the activity of protoscoleces of Echinococcus granulosus in vitro. Methods protoscoleces of Echinococcus granulosus was divided into six groups and cultured in vitro.AZD2014 was added into the four of six groups to make their concentration as 200μmol/L, 100μmol/L, 50μmol/L and25μmol/L respectively. The vitality of the protoscoleces were studied under an optical microscope by using 0.1% eosin stain for10 days. The experiment was repeated three times, and the protoplasma activity curve was drawn; the expression of p-Akt1, p-4 EBP1, p-S6 K1 which were extracted from the protoscoleces that had been cultured in 100μmol/L of AZD2014 for 4 days were detected by using Western-blot; the activity of Caspase-3 which were extracted from the protoscoleces that had been cultured in 100μmol/L of AZD2014 for 4 days was extracted according to the instructions of the Caspase-3 kit. R esults after treated with 100μmol/L AZD2014 for 4 days, the protoscoleces＇ activity began to decline and the survival rate was（75.93%±7.28%）. After being cultured in AZD2014 for 10 days, the activity of protoscoleces was only（24.03%±1.01%） in 100μmol/L and the value was（2.40%±1.14%） in 200μmol/L; The expression of p-Akt1, p-4 EBP1 and p-S6 K1 decreased after culturing protoscoleces in 100μmol/L of AZD2014 for 4 days; The expression of Caspase-3 increased after culturing protoscoleces in100 μmol/L of AZD2014 for 4 days. Conclusion It can be concluded that AZD2014 has the ability of anti-protoscoleces,which could be a new drug to against Echinococcus granulosus in vitro in theory.