中缅边境恶性疟原虫对青蒿素类药体外敏感性检测和K-13基因突变的相关性研究  

In vitro susceptibility of Plasmodium falciparumto artemisinin drugs and K13-propeller polymorphisms at the China-Myanmar border

作　　者：张艳梅[1,2,3] 吴艳瑞[4] 胡月[5] 王丽琼[1] 阮永华[5] 马宁[1] 李思熳[6] 王颖娜[1] 贾丹丹[1] 向征[1] 杨照青[1] 

机构地区：[1]昆明医科大学病原微生物系与免疫学系,昆明650500 [2]昆明医科大学第一附属医院感染内科,昆明650500 [3]云南公共卫生与疾病防控协同创新中心,昆明650031 [4]昆明医科大学基础医学院细胞生物学与医学遗传学系,昆明650500 [5]昆明医科大学病理与病理生理学系,昆明650500 [6]昆明医科大学基础医学院生物化学与分子生物学系,昆明650500

出　　处：《中国人兽共患病学报》2016年第3期219-223,共5页Chinese Journal of Zoonoses

基　　金：国家自然科学基金(No.U1202226;81161120421;31260508);云南省科技厅-昆明医科大学应用基础研究联合专项(No.2015FB034;2014FB005);昆明医科大学创新基金(2014-D2)联合资助;云南公共卫生与疾病防控协同创新中心项目(No.2014YNPHXT05);云南省自然科学基金(No.2012FB153)~~

摘　　要：目的研究中缅边境恶性疟原虫对青蒿素类药物抗药机制。方法收集分离自2008—2010年中缅边境确诊为恶性疟病人体内的疟原虫,共34株,进行体外培养;提取原虫血DNA,采用聚合酶链式反应(PCR)方法,扩增PF3D7-1343700(K-13)基因,对目标基因进行全基因测序,检测K-13基因变异情况;依据基因有无变异分为两组,对比两个组别疟原虫对3种抗疟药青蒿琥酯(Artesunate,AS)、双氢青蒿素(Dihydroartemisinine,DHA)和青蒿素(artemisinin,ART)的半效抑虫浓度(IC50值),并与野生株3D7做对比;随后将这些疟原虫在环状体时期暴露于大剂量的DHA下,6h后洗尽药物,再经过66h培养后涂片,计算生存率(RSA),比较变异株和非变异株之间有无差异。结果检测发现疟原虫K13基因存在137-142位点NN插入(占82.4%),另有E252Q、F446I、C469Y、R539T、P553L、P574L、H719N位点突变,均伴有NN插入,其中F446I(占44.1%)。病人株的IC50值和野生株无统计学差异(P>0.05),但病人株的RSA高于野生株(P<0.05);有K13基因变异组和无变异组对AS、DHA、ART的IC50值无统计学差异(P>0.05),但有基因变异组的RSA高于无基因变异组(P<0.05)。结论本研究结果发现K-13基因变异与恶性疟原虫对青蒿素类药物抗药性可能相关。This study aims to evaluate drug resistance mechanism to artemisinin derivatives in Plasmodium falciparum.The parasites of patients collected from the China-Myanmar border from 2008 to 2010were cultured in vitro,and DNAs were extracted.The full-length of K13 gene was sequenced in all samples using polymerase chain reaction and direct sequencing methods.To measure drug susceptibilities in artesunate（AS）,dihydroartemisinin（DHA）and artemisinin（ART）,we calculated the50%inhibitory concentrations（IC50s）.Parasites were exposed to high-dose of dihydroartemisinin in ring stage for 6hours,and the survival rates of the ring stage（RSA）was calculated after 66 hours.We found that there were no difference in IC50 s in patient isolates and Standard strain-3D7,and RSA in parasite isolates was significantly higher than 3D7.Sever point mutations（E252Q F446IC469 Y R539T P553 L P574L H719N）were identified in58.8% of the parasite isolates,and F446 Imutation predominated in 44.1% of the parasite isolates.NN-insert mutation predominated in 82.4%,the total point mutations were accompanied by NN-insert.All sample were divided into two groups：gene mutation and no-mutation.We found that there were no difference in IC50 in gene mutation and no-mutation in K-13,and RSA in parasite isolates with K13 mutations was significantly higher than that in wild-type isolates.This result suggest that K13 mutations were possible correlated with artemisinin resistance.

关 键 词：恶性疟原虫 青蒿素类药物 耐药机制 PF3D7_1343700 

分 类 号：R382.3[医药卫生—医学寄生虫学]