湿热证鼠疟伯氏疟原虫K173抗性培育及相关基因多态性研究

Malaria is a serious disease that endanger human health. In recent years, mosquito-borne distribution area extends as the global becomes warming, and the trend of control malaria still pessimism. In the aspect of disease, death and economic loss, malaria becomes the most serious of insect-borne infectious diseases in the world. Guided by the theory of traditional of Chinese medicine, this study adopts complex factors, simulates of natural infection way, synthesizes environmental factors, habitus, constitution, and pathogenic microorganism to establish the malaria model with pattern of damp-heat, cultivates cell lines of Artemisinin group (including the resistance strain of Artemisinin and the normal) and Piperaquine group (including the resistance strain of Piperaquine and the normal) with pattern of damp-heat. With the nested PCR will be used to amplify of gene fragment and sequencing will be analyzed by Mega software for analysis of the mutation frequency of each drug resistance related sites. Thereafter, we may use the linkage disequilibrium analysis and genetically modified method to determine the resistance genes, then, compare between Artemisinin and Piperaquine in two different environment on the effect of resistance will be made.This study is to evaluatethe correlation of resistance of compound artemisinin and damp-heat environment, explorethe mechanism of resistance of artemisinin production, provide the evidence to prolong the usage of the compound artemisinin in Asia and Africa malaria area with damp-heat environment, grasp the rule of the regional environment of p. falciparum resistance to drugs, and analyze the resistance and gene polymorphism of p. falciparum under the presence of the malaria parasite drug. Victims of malaria people will enjoy the traditional Chinese medicine to bring the Gospel in China.

疟疾是一种严重危害人体健康的疾病。近年来，全球气候变暖,蚊媒分布区域扩大,疟疾仍无递减趋势, 成为世界上在疾病、死亡和经济损失方面最严重的虫媒传染病。本研究以中医理论为指导,采用复合因素,模拟自然感染方式,综合环境气候、体质及病原微生物等因素建立合适的鼠疟湿热证动物模型；培育出湿热证青蒿素和哌喹抗性株组和常态组，采用巢式PCR扩增全长基因，测序后以Mega软件分析比对，分析各抗药性相关位点的突变频率；并利用连锁不平衡分析、转基因方法寻找确定耐药基因；比较两种不同环境对青蒿素、哌喹耐药性的影响。评价复方青蒿素抗性与湿热环境的相关性，探索青蒿素抗性产生的机制，为进一步延缓复方青蒿素在亚非洲湿热环境疟区使用寿命提供依据；掌握区域环境恶性疟原虫对药物抗性的影响，分析恶性疟原虫在药物压力下是否存在疟原虫抗药性及基因多态性，让受疟疾危害的人民享受到中国中医药带来的福音。

疟疾是一种严重危害人体健康的疾病，近年来，全球气候变暖，蚊媒分布区域扩大，疟疾仍无递减趋势，成为世界上在疾病、死亡和经济损失方面最严重的虫媒传染病。本研究以中医理论为指导，采用复合因素，模拟自然感染方式，综合环境气候、体质及病原微生物等因素建立了病证结合的鼠疟湿热证动物模型。. 培育出湿热证青蒿素和哌喹抗性株组和常态组，使用小剂量递增的方法，在持续的药物压力下，伯氏疟原虫K173株对哌喹可以产生重度抗性，但对青蒿素的抗性则起伏波动，较难产生重度抗性。湿热证小鼠和湿热环境小鼠在感染早期血清IFN－γ水平和IFN－γ/IL-4比值比正常环境小鼠要高，差异有统计学意义（P<0.05）。IFN－γ因子可能激活并增强巨噬细胞吞噬功能抑制疟原虫生长，但后期IFN－γ、IL-4因子大量减少，巨噬细胞吞噬活性降低，原虫感染率爆发性增长，最终导致小鼠死亡。. 分别对伯氏疟原虫K173的青蒿素敏感株及其抗性株的Kelch 13基因片段的进行特异性扩增和测序，研究结果显示：与敏感株相比，抗性株共有义突变10处，分别Y43F、I63M、N104S、A121T、N123S、S133N、N155S、E177D、S548L以及P723S。然而，尚未检测到与东南亚地区恶性疟原虫青蒿素耐药株密切相关的K13基因突变点，如C458Y，r539t，y493h，m476i。研究结果提示，在青蒿素类药物长期胁迫下，伯氏疟虫k173株的k13基因同样出现较多的突变，但与东南亚恶性疟原虫青蒿素耐药株的k13基因突变位点有差异，需要进一步的研究确认何处突变位点与青蒿素抗性有关。 . 青蒿素及其衍生物作为抗疟一线用药，具有高效，快速及毒副作用小的特点，它的发现和使用使疟疾的流行得到了一定程度的遏制。但任何一类抗疟药多次长期使用最终都会导致抗药性的出现。关于恶性疟原虫对青蒿素抗药性机制的研究，对全球消灭疟疾的工作具有深远的意义与影响。