影响约氏疟原虫在按蚊体内发育的因素及机制的初步研究

发布时间：2018-01-01 20:38

本文关键词：影响约氏疟原虫在按蚊体内发育的因素及机制的初步研究　出处：《第三军医大学》2011年硕士论文　论文类型：学位论文

【摘要】：疟疾是世界上危害最为严重的传染病,该病每年感染1亿多人并造成80多万人死亡。目前,疟疾的防治措施主要是服用抗疟药物及避免或减少蚊虫的叮咬如使用驱避剂或浸药蚊帐等。然而单纯以药物治疗为主要策略,难以达到在全世界范围内消除疟疾的目标。因此,阻断疟疾传播成为控制和消灭全球疟疾的可靠途径和迫切需要。阻断疟疾传播的方法之一是依赖传播阻断疫苗(transmission?blocking vaccines,TBVs)的应用。虽然TBVs的研制已取得阶段性的进展,但是由于抗原的免疫原性弱、佐剂的应用效果不理想及抗原不恰当折叠等因素产生的影响,阻碍了疟疾TBVs的发展,这就迫切需要一种新的阻断传播的策略。阻断传播的前提是对疟原虫—蚊媒相互关系的深入认识和了解。在按蚊-疟原虫相互关系的研究中发现蚊的天然免疫是其抗疟原虫感染的主要机制。近期研究发现,采用抗生素药物抑制蚊肠道菌群后可以导致冈比亚按蚊免疫反应下调,从而促进疟原虫在蚊体内的发育;氯喹导致蚊丝氨酸蛋白酶、抗菌肽表达下调,影响按蚊丝氨酸蛋白酶级联反应,干扰信号转导和抗菌肽转录水平,从而增加了按蚊对疟原虫的易感性,有利于按蚊对疟原虫的传播。我们的前期研究发现抗疟药物-硝喹能诱导斯氏按蚊对约氏疟原虫的黑化,启动按蚊对疟原虫的免疫反应,从而阻断疟原虫在蚊体内的发育,提示诸多干预因素可以调节按蚊免疫系统,影响疟原虫在按蚊体内的发育。本课题利用大劣按蚊/约氏疟原虫不易感模型和斯氏按蚊/约氏疟原虫易感模型,对各种不同因素影响约氏疟原虫在按蚊体内发育及其机制进行初步探讨。 1.肠道菌群、TEP1对约氏疟原虫在大劣按蚊体内发育影响及其机制初步研究利用约氏疟原虫/大劣按蚊不易感蚊模型,通过抗生素抑制蚊肠道菌群后,结果显示给抗生素感染组蚊感染卵囊数明显高于正常感染组蚊感染卵囊数(P0.0001),卵囊感染度增加7-10倍,感染率增加近1倍,说明抗生素抑制蚊肠道菌群导致约氏疟原虫对大劣按蚊的易感度大大增加,提示肠道菌群在疟原虫感染大劣按蚊的过程中起着重要的作用。 RT-PCR结果显示抗生素处理肠道菌群后,使含硫脂蛋白1( thioester-containing proteins1 , TEP1) cDNA的转录水平明显下调,提示肠道菌群可能通过调节TEP1的表达影响约氏疟原虫在大劣按蚊体内的发育。我们采用RT-PCR方法,观察肠道菌群未经处理情况下,检测大劣按蚊TEP1 cDNA在正常吸糖水、吸正常血和感染疟原虫三种情况下的转录变化,结果显示吸血和感染疟原虫均可诱导TEP1 cDNA的转录上调,其中感染疟原虫明显上调TEP1 cDNA的转录表达,然而在清除肠道菌群后,疟原虫感染却无法明显上调TEP1 cDNA的表达,提示肠道菌群可能通上调TEP1激活抗疟免疫反应,抑制疟原虫在按蚊体内发育,提示TEP1可能作为一重要效应因子参与肠道菌群激活的基础免疫反应。肠道菌群未处理情况下,本实验对按蚊TEP1进行RNAi,结果显示大劣按蚊蚊胃中约氏疟原虫的卵囊数量增加10-15倍,感染率增加1倍,说明在缺少TEP1的情况下,肠道菌群无法抑制约氏疟原虫在大劣按蚊体内的发育,提示在肠道菌群抑制疟原虫发育的过程中TEP1是必不可少的一个环节。本研究提示按蚊肠道菌群可以通过调节TEP1的表达抑制疟原虫在大劣按蚊体内的发育;TEP1作为重要的免疫反应因子可能参与按蚊基础免疫反应,维持按蚊肠道菌群在正常水平。然而,当疟原虫入侵后引起肠道菌群变化刺激上调TEP1表达,识别并结合疟原虫启动杀伤疟原虫机制。对肠道菌群、TEP1影响约氏疟原虫在按蚊体发育的机制的研究,可以改变按蚊对疟原虫的易感性,可能为阻断疟疾在蚊期传播提供一新的切入点。 2、蒿甲醚影响约氏疟原虫在斯氏按蚊体内发育及其机制研究利用约氏疟原虫/斯氏按蚊易感模型,斯氏按蚊吸饲蒿甲醚糖水,结果发现吸饲蒿甲醚糖水斯氏按蚊体内的卵囊数量呈3-5倍的增加,说明蒿甲醚可以促进并有利于约氏疟原虫雌雄配子体在斯氏按蚊体内向卵囊方向的发育。 RT-PCR结果显示蒿甲醚明显抑制了斯氏按蚊一氧化氮合成酶(NO synthase, NOS),TEP1和前酚氧化酶(Prophenoloxidase,PPO)三个重要的蚊抗疟原虫免疫相关基因的表达,提示蒿甲醚可能通过抑制NO合成和黑化包被反应或是裂解反应,致使斯氏按蚊对疟原虫免疫反应减弱,促进疟原虫在按蚊体内更好的发育。本研究通过对影响约氏疟原虫在按蚊体内发育的因素及其机制的初步探讨,如何降低疟原虫对媒介的易感性为阻断疟疾的传播提供更多的理论依据,为研制新的、高效的阻断传播手段提供一个新的切入点。同时也提示,广泛应用于临床和现场杀红内期药物蒿甲醚反而可以促进按蚊感染疟原虫能力提高,有利于疟疾的流行和扩散。这将对如何控制疟疾流行、媒介传播提出了新的问题,如何合理使用青蒿素类杀红内期药物提出了新的研究课题。
[Abstract]:Malaria is the world the most serious hazards of infectious diseases, the disease each year infected more than 100 million people and killed about 800000 people. At present, the malaria control measures are mainly taking anti malaria drugs and avoid or reduce mosquito bites as repellents or impregnated bednets. However the drug treatment is the main strategy, it is difficult to to achieve the goal of eliminating malaria worldwide. Therefore, blocking the spread of malaria as a reliable way to control and eliminate the global malaria and urgent need. One method of blocking the spread of malaria is dependent on the transmission blocking vaccine (transmission? Blocking vaccines, TBVs) application. Although the development of TBVs has made gradual progress, but because of antigen immunogenicity is weak, the impact effect is not ideal adjuvant and antigen improper folding and other factors, hinder the development of malaria TBVs, there is an urgent need for a new resistance The strategy of breaking communication.
Blocking transmission is the premise of in-depth knowledge and understanding of the relationship between the malaria mosquito, Anopheles. In the study of the relationship between the parasite - found that naturalimmune response is the main mechanism against Plasmodium infection. A recent study found that inhibiting the activity of intestinal flora by antibiotics drugs can lead to lowered Gambia Anopheles immune response, so as to promote the parasites in mosquitoes mosquito development; chloroquine resulted serine protease, antimicrobial peptide expression, effect of Anopheles serine protease cascade, and antibacterial peptide transcription interference signal transduction, thereby increasing the susceptibility of Anopheles to Plasmodium, is conducive to the spread of malaria Anopheles. Our preliminary study showed that the antimalarial drug NITROQUINE induced melanization of Plasmodium yoelii in Anopheles stephensi, initiating the immune response of Anopheles Plasmodium parasites in mosquitoes, thereby blocking the development. In many intervention factors can regulate the immune system in Anopheles mosquitoes, the development of Plasmodium. The subject of the use of Anopheles dirus Plasmodium yoelii model and not susceptible to Plasmodium yoelii / Anopheles stephensi susceptibility model of different kinds of influence factors of Plasmodium yoelii in Anopheles in vivo and its mechanism.
1. intestinal flora, the effect of TEP1 on the development of Anopheles de Anopheles and its mechanism
The use of Plasmodium yoelii / Anopheles dirus not susceptible mosquito model, inhibitory activity of intestinal flora by antibiotics, according to the result of antibiotic infection group was significantly higher than the normal number of oocysts infected mosquito mosquito infection infection group (P0.0001), the number of oocysts of oocysts increased 7-10 times, the infection rate increased nearly 1 times that of antibiotics to inhibit the mosquito the intestinal microflora causes of Plasmodium yoelii in Anopheles dirus susceptible degree is greatly increased, intestinal flora in Anopheles dirus Plasmodium infection plays an important role.
RT-PCR results showed that the transcription level of sulfur containing lipoprotein 1 (thioester-containing proteins1, TEP1) cDNA was down regulated after antibiotic treatment of intestinal microflora, suggesting that intestinal microflora may affect the development of Plasmodium falciparum in the Anopheles DUS.
We use RT-PCR method, observe the intestinal flora of untreated cases, detection of Anopheles dirus TEP1 cDNA in normal suction suction syrup, normal blood and infected with the parasite transcriptional changes under three kinds of situations, the results show the increased transcription of blood and infected with the parasite could induce TEP1 cDNA, which infected transcription of TEP1 were up-regulated the expression of cDNA however, in the removal of intestinal flora after Plasmodium infection but not significantly up-regulated the expression of TEP1 cDNA, suggesting that the gut microbiota may be through upregulation of TEP1 activation of anti malarial immune response, inhibiting the development of parasites in mosquitoes, suggesting that TEP1 may act as an important effect factor in the intestinal flora based immune activation reaction.
The intestinal flora of untreated cases, the experiments for RNAi of Anopheles TEP1, showed increased 10-15 times the number of oocysts of Plasmodium yoelii in Anopheles dirus mosquitoes in the stomach, the infection rate increased 1 times, in the absence of TEP1, intestinal bacteria can inhibit Plasmodium yoelii in inferior by mosquitoes development in the process, suggesting that intestinal bacteria inhibit Plasmodium development in TEP1 is an indispensable link.
This study suggests that the intestinal flora of Anopheles dirus Plasmodium can inhibit in vivo growth by regulating the expression of TEP1; TEP1 as an important factor may be involved in the immune response of Anopheles based immune response, maintain Anopheles gut flora in normal level. However, when the change in intestinal flora caused by Plasmodium invasion after stimulated upregulation of TEP1 expression and recognition with the start of Plasmodium anti parasite mechanism. On intestinal microflora, studies in the mechanism of the development of the Anopheles TEP1 affect Plasmodium yoelii Anopheles can change on Plasmodium susceptibility, may be blocking malaria in mosquitoes spread to provide a new starting point.
2, artemether affects the development of Plasmodium J. in the body of Anopheles sinensis and its mechanism
The use of Plasmodium yoelii / Anopheles stephensi model of Anopheles stephensi suction feeding artemether syrup, the absorption quantity of artemether in feeding Anopheles stephensi oocysts was increased by 3-5 times, that artemether can promote and facilitate the male and female gametophyte of Plasmodium yoelii by mosquitoes in the STEVENSI direction within oocysts the development.
RT-PCR results showed that Anopheles stephensi nitric oxide synthase was inhibited by artemether (NO synthase, NOS, TEP1) and prophenoloxidase (Prophenoloxidase, PPO) expression of three important anti Plasmodium mosquito immune related genes, suggesting that artemether may inhibit NO synthesis and blackening coated reaction or cracking reaction, resulting in the STEVENSI Anopheles immune response to the parasite weakens, promote the development of parasites in mosquitoes better.
Based on the influence of Plasmodium yoelii in Anopheles in vivo development to discuss the factors and mechanism, how to reduce the susceptibility of Plasmodium media to provide more theoretical basis for blocking the spread of malaria, for the development of new, efficient means to prevent the spread of a new starting point. At the same time also suggested that, widely used in clinical and the site of the kill erythrocytic drugs artemether but can promote Anopheles infected ability are popular and conducive to the diffusion of malaria. It will be on how to control malaria, the media presents new problems, how to make rational use of artemisinin drugs kill erythrocytic stage provides a new research topic.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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