蚊媒在恶性疟原虫抗药性选择传播中的作用及机制研究

Plasmodium falciparum drug resistance has become one of the most important barriers to malaria treatment and control, in addition to monitoring the Plasmodium falciparum drug resistance varation in human body, the selective spread of drug-resistant malaria parasite by the mosquito-borne was unclear. Recent studies have shown that in the vector selection pressures have an effect on pathogens and lead to phenotypic changes; Preliminary study found that chloroquine and mefloquine resistance of the plasmodium were changed by the mosquito-borne and the chloroquine resistance was increased, the mefloquine resistance was lost; Anopheles innate immunity may play an important role on this regard. Therefore, based on the preliminary studies, in this project we will use PCR-RFLP to observe Plasmodium falciparum resistance gene (pfCRT, pfMDR1,pfDHFR) polymorphism from human and Anopheles in malaria-endemic areas, analyze of the mutation site and ways and SNP gene frequency, and the choice sites on Plasmodium by the Anopheles;then separate Plasmodium falciparum resistance single strains to be directly infected Anopheles, and combine with RNAi to suppress expression of TEP1 and observe Plasmodium falciparum resistance gene change by the mosquito-borne, so as to explore the role and mechanism of the mosquito-borne Plasmodium falciparum resistance selective transmission. This helps to develop effective blocking resistance transmission measures, and also to predict and control the Plasmodium falciparum drug resistance transmission.

恶性疟原虫药物抗性已成为疟疾治疗和控制的重要障碍之一,而目前实施的药物抗性监控仅关注人体内疟原虫抗性变化,对于抗性经蚊媒传播遗传变化研究较少。前期研究发现鼠约氏疟原虫氯喹和甲氟喹抗性经蚊传后氯喹抗性增加,甲氟喹抗性丢失;而按蚊天然免疫可能对此发挥重要作用。因此本课题拟在前期研究基础上,首先利用PCR-RFLP分析疟疾流行现场的人和按蚊体内恶性疟原虫抗氯喹或磺胺相关基因(pfCRT、pfMDR1或pfDHFR)多态性,分析突变位点和方式及SNP基因频率等,并分析媒介按蚊对疟原虫可能的选择位点；然后分离抗性单虫株直接感染按蚊,并结合RNAi技术抑制按蚊重要免疫识别基因(TEP1等)表达,观察抗药相关基因经免疫缺陷蚊媒传播有无遗传变化,从而探讨蚊媒在恶性疟原虫抗药性选择性传播中的作用和机制。这有助于制定有效的阻断抗性传播措施，还为预测和控制恶性疟原虫药物抗性传播扩散提供新的思路。

恶性疟原虫药物抗性已成为疟疾治疗和控制的重要障碍之一,而目前实施的药物抗性监控仅关注人体内疟原虫抗性变化,对于抗性经蚊媒传播遗传变化研究较少。前期研究发现鼠约氏疟原虫氯喹和甲氟喹抗性经蚊传后氯喹抗性增加,甲氟喹抗性丢失;而按蚊天然免疫可能对此发挥重要作用。因此本课题拟在前期研究基础上,首先分析通过分析疟疾流行现场恶性疟原虫抗性基因多态性差异，显示恶性疟原虫DHFR基因双重突变虫株较仅出现 S108N单位点突变的恶性疟原虫株抗性水平高，并且三重突变较双重突变虫株的 IC50高，因此说明该地区的恶性疟原虫DHFR基因突变程度较以往有所升高，提示恶性疟原虫对乙胺嘧啶的抗性尚无减弱趋势。对PfDHPS的多态性进行比较，本研究仅发现三突变型（SGEG、SGNG和AGEA），其中以AGEA型所占比例较高，达50%。因此说明该区域恶性疟原虫DHPS基因突变程度与以往相比无明显变化。对PfMDR1的多态性分析显示，本组标本中主要为N86Y的突变，频率达32%，而2007-2009年的突变频率均在10%以下。非洲、亚洲等地的体外实验及临床研究结果显示，PfMDR1分子在86位点的突变与氯喹抗性密切相关。提示中缅边境地区恶性疟原虫DHFR和MDR1基因突变升高，而DHPS和ATPase6基因突变程度无明显变化，可为恶性疟的防治提供参考。 其次通过观察抗疟药对按蚊天然免疫影响以及媒介按蚊天然免疫对疟原虫抗性株的影响发现疟原虫抗性株感染按蚊后13d，可以明显下调按蚊的重要天然免疫基因，提示按蚊的天然免疫（如JNK信号通路、补体途径、黑化反应等）可能影响蚊体内疟原虫抗性株的抗性表型变化以及抗性株的发育增殖，抗性株疟原虫可能逃避按蚊天然免疫，进而在疟原虫抗性传播和筛选中发挥重要作用；同时发现小鼠体内的某些免疫成分可以诱导按蚊天然免疫从而抑制疟原虫在蚊体内的发育。提示抗性虫株在按蚊体内的发育及抗性表型变化可能与感染按蚊的天然免疫及小鼠的免疫血清成分相关。