实时荧光定量PCR测定瑞香素类抗疟化合物对恶性疟原虫核糖核酸还原酶基因表达的影响  

Effect of daphnetin and its derivates on gene expression of ribonucleotide reductase from Plasmodium falciparum in vitro detected by real-time PCR

作　　者：黄芳[1] 汤林华[1] 陈博[2] 周水森[1] 王琴美[1] 

机构地区：[1]中国疾病预防控制中心寄生虫病预防控制所,世界卫生组织疟疾、血吸虫病和丝虫病合作中心,卫生部寄生虫病原与媒介生物学重点实验室,上海200025 [2]复旦大学生命科学学院遗传系,上海200433

出　　处：《中国病原生物学杂志》2008年第4期288-291,共4页Journal of Pathogen Biology

摘　　要：目的测定瑞香素类抗疟化合物对恶性疟原虫核糖核酸还原酶（RNR）基因表达的影响。方法体外同步培养的恶性疟原虫经瑞香素及其衍生物DA78与DA79作用后,抽提总RNA,并合成相应的特异性引物,运用实时荧光定量聚合酶链反应（Real-time PCR）测定瑞香素类抗疟化合物对恶性疟原虫RNR基因表达的影响。结果恶性疟原虫体外经瑞香素及其衍生物作用后的RNR基因扩增标准曲线在10^5～10^9拷贝（copies）/ml范围内呈良好的线性关系,R^2为0.997 52;瑞香素及其衍生物DA78与DA79对恶性疟原虫RNR作用后,其原始拷贝数分别为81 173.23、124 830.83和74 801.35,与对照组比较差异有统计学意义（P〈0.01）,而瑞香素的铁螯合能力被饱和后,对该基因的表达未产生显著抑制（P〉0.01）。结论瑞香素类抗疟化合物体外抑制恶性疟原虫RNR的基因表达,RNR可能作为瑞香素类抗疟化合物抗疟作用的候选靶分子之一。Objective To determine the effect of daphnetin and its derivates on gene expression of ribonucleotide reductase（RNR） from Plasmodium falciparum.Methods P.falciparum synchronized with sorbitol in vitro was treated by daphnetin and its derivates,and RNA was extracted and special gene primer was designed and synthesized.The gene express level of daphnetin and its derivates on RNR was tested by real-time PCR.Results The standard curves of the amplification products of RNR were created.Their standard curves showed linear relationship at the range of 10^5-10^9 copies/ml and the R^2 values were 0.997 52.There was the inhibition effect on expression of P.falciparum RNR transcripts by daphnetin,DA78 and DA79.And the original copies was 81 173.23,124 830.83,74 801.35,respectively,compared with the control group,which showed significant difference（n=3,P〈0.01）.No significant inhibition effects on expression of these three genes when the chelator had been saturated with iron.Conclusion The gene express level of daphnetin and its derivates on RNR was inhibited and RNR may be one of the molecular targets of antimalarial activity.

关 键 词：瑞香素 疟原虫 恶性 核糖核酸还原酶 实时荧光定量聚合酶链反应 

分 类 号：R382.31[医药卫生—医学寄生虫学]