本文關(guān)鍵詞：影響約氏瘧原蟲(chóng)在按蚊體內(nèi)發(fā)育的因素及機(jī)制的初步研究　出處：《第三軍醫(yī)大學(xué)》2011年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 約氏瘧原蟲(chóng) 大劣按蚊 RT- PCR 含硫酯鍵蛋白1 ( thioester-containing proteins TEP1) 免疫反應(yīng) 腸道菌群 斯氏按蚊 蒿甲醚

【摘要】：瘧疾是世界上危害最為嚴(yán)重的傳染病,該病每年感染1億多人并造成80多萬(wàn)人死亡。目前,瘧疾的防治措施主要是服用抗瘧藥物及避免或減少蚊蟲(chóng)的叮咬如使用驅(qū)避劑或浸藥蚊帳等。然而單純以藥物治療為主要策略,難以達(dá)到在全世界范圍內(nèi)消除瘧疾的目標(biāo)。因此,阻斷瘧疾傳播成為控制和消滅全球瘧疾的可靠途徑和迫切需要。阻斷瘧疾傳播的方法之一是依賴傳播阻斷疫苗(transmission?blocking vaccines,TBVs)的應(yīng)用。雖然TBVs的研制已取得階段性的進(jìn)展,但是由于抗原的免疫原性弱、佐劑的應(yīng)用效果不理想及抗原不恰當(dāng)折疊等因素產(chǎn)生的影響,阻礙了瘧疾TBVs的發(fā)展,這就迫切需要一種新的阻斷傳播的策略。 阻斷傳播的前提是對(duì)瘧原蟲(chóng)—蚊媒相互關(guān)系的深入認(rèn)識(shí)和了解。在按蚊-瘧原蟲(chóng)相互關(guān)系的研究中發(fā)現(xiàn)蚊的天然免疫是其抗瘧原蟲(chóng)感染的主要機(jī)制。近期研究發(fā)現(xiàn),采用抗生素藥物抑制蚊腸道菌群后可以導(dǎo)致岡比亞按蚊免疫反應(yīng)下調(diào),從而促進(jìn)瘧原蟲(chóng)在蚊體內(nèi)的發(fā)育;氯喹導(dǎo)致蚊絲氨酸蛋白酶、抗菌肽表達(dá)下調(diào),影響按蚊絲氨酸蛋白酶級(jí)聯(lián)反應(yīng),干擾信號(hào)轉(zhuǎn)導(dǎo)和抗菌肽轉(zhuǎn)錄水平,從而增加了按蚊對(duì)瘧原蟲(chóng)的易感性,有利于按蚊對(duì)瘧原蟲(chóng)的傳播。我們的前期研究發(fā)現(xiàn)抗瘧藥物-硝喹能誘導(dǎo)斯氏按蚊對(duì)約氏瘧原蟲(chóng)的黑化,啟動(dòng)按蚊對(duì)瘧原蟲(chóng)的免疫反應(yīng),從而阻斷瘧原蟲(chóng)在蚊體內(nèi)的發(fā)育,提示諸多干預(yù)因素可以調(diào)節(jié)按蚊免疫系統(tǒng),影響瘧原蟲(chóng)在按蚊體內(nèi)的發(fā)育。本課題利用大劣按蚊/約氏瘧原蟲(chóng)不易感模型和斯氏按蚊/約氏瘧原蟲(chóng)易感模型,對(duì)各種不同因素影響約氏瘧原蟲(chóng)在按蚊體內(nèi)發(fā)育及其機(jī)制進(jìn)行初步探討。 1.腸道菌群、TEP1對(duì)約氏瘧原蟲(chóng)在大劣按蚊體內(nèi)發(fā)育影響及其機(jī)制初步研究 利用約氏瘧原蟲(chóng)/大劣按蚊不易感蚊模型,通過(guò)抗生素抑制蚊腸道菌群后,結(jié)果顯示給抗生素感染組蚊感染卵囊數(shù)明顯高于正常感染組蚊感染卵囊數(shù)(P0.0001),卵囊感染度增加7-10倍,感染率增加近1倍,說(shuō)明抗生素抑制蚊腸道菌群導(dǎo)致約氏瘧原蟲(chóng)對(duì)大劣按蚊的易感度大大增加,提示腸道菌群在瘧原蟲(chóng)感染大劣按蚊的過(guò)程中起著重要的作用。 RT-PCR結(jié)果顯示抗生素處理腸道菌群后,使含硫脂蛋白1( thioester-containing proteins1 , TEP1) cDNA的轉(zhuǎn)錄水平明顯下調(diào),提示腸道菌群可能通過(guò)調(diào)節(jié)TEP1的表達(dá)影響約氏瘧原蟲(chóng)在大劣按蚊體內(nèi)的發(fā)育。 我們采用RT-PCR方法,觀察腸道菌群未經(jīng)處理情況下,檢測(cè)大劣按蚊TEP1 cDNA在正常吸糖水、吸正常血和感染瘧原蟲(chóng)三種情況下的轉(zhuǎn)錄變化,結(jié)果顯示吸血和感染瘧原蟲(chóng)均可誘導(dǎo)TEP1 cDNA的轉(zhuǎn)錄上調(diào),其中感染瘧原蟲(chóng)明顯上調(diào)TEP1 cDNA的轉(zhuǎn)錄表達(dá),然而在清除腸道菌群后,瘧原蟲(chóng)感染卻無(wú)法明顯上調(diào)TEP1 cDNA的表達(dá),提示腸道菌群可能通上調(diào)TEP1激活抗瘧免疫反應(yīng),抑制瘧原蟲(chóng)在按蚊體內(nèi)發(fā)育,提示TEP1可能作為一重要效應(yīng)因子參與腸道菌群激活的基礎(chǔ)免疫反應(yīng)。 腸道菌群未處理情況下,本實(shí)驗(yàn)對(duì)按蚊TEP1進(jìn)行RNAi,結(jié)果顯示大劣按蚊蚊胃中約氏瘧原蟲(chóng)的卵囊數(shù)量增加10-15倍,感染率增加1倍,說(shuō)明在缺少TEP1的情況下,腸道菌群無(wú)法抑制約氏瘧原蟲(chóng)在大劣按蚊體內(nèi)的發(fā)育,提示在腸道菌群抑制瘧原蟲(chóng)發(fā)育的過(guò)程中TEP1是必不可少的一個(gè)環(huán)節(jié)。 本研究提示按蚊腸道菌群可以通過(guò)調(diào)節(jié)TEP1的表達(dá)抑制瘧原蟲(chóng)在大劣按蚊體內(nèi)的發(fā)育;TEP1作為重要的免疫反應(yīng)因子可能參與按蚊基礎(chǔ)免疫反應(yīng),維持按蚊腸道菌群在正常水平。然而,當(dāng)瘧原蟲(chóng)入侵后引起腸道菌群變化刺激上調(diào)TEP1表達(dá),識(shí)別并結(jié)合瘧原蟲(chóng)啟動(dòng)殺傷瘧原蟲(chóng)機(jī)制。對(duì)腸道菌群、TEP1影響約氏瘧原蟲(chóng)在按蚊體發(fā)育的機(jī)制的研究,可以改變按蚊對(duì)瘧原蟲(chóng)的易感性,可能為阻斷瘧疾在蚊期傳播提供一新的切入點(diǎn)。 2、蒿甲醚影響約氏瘧原蟲(chóng)在斯氏按蚊體內(nèi)發(fā)育及其機(jī)制研究 利用約氏瘧原蟲(chóng)/斯氏按蚊易感模型,斯氏按蚊吸飼蒿甲醚糖水,結(jié)果發(fā)現(xiàn)吸飼蒿甲醚糖水斯氏按蚊體內(nèi)的卵囊數(shù)量呈3-5倍的增加,說(shuō)明蒿甲醚可以促進(jìn)并有利于約氏瘧原蟲(chóng)雌雄配子體在斯氏按蚊體內(nèi)向卵囊方向的發(fā)育。 RT-PCR結(jié)果顯示蒿甲醚明顯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)三個(gè)重要的蚊抗瘧原蟲(chóng)免疫相關(guān)基因的表達(dá),提示蒿甲醚可能通過(guò)抑制NO合成和黑化包被反應(yīng)或是裂解反應(yīng),致使斯氏按蚊對(duì)瘧原蟲(chóng)免疫反應(yīng)減弱,促進(jìn)瘧原蟲(chóng)在按蚊體內(nèi)更好的發(fā)育。 本研究通過(guò)對(duì)影響約氏瘧原蟲(chóng)在按蚊體內(nèi)發(fā)育的因素及其機(jī)制的初步探討,如何降低瘧原蟲(chóng)對(duì)媒介的易感性為阻斷瘧疾的傳播提供更多的理論依據(jù),為研制新的、高效的阻斷傳播手段提供一個(gè)新的切入點(diǎn)。同時(shí)也提示,廣泛應(yīng)用于臨床和現(xiàn)場(chǎng)殺紅內(nèi)期藥物蒿甲醚反而可以促進(jìn)按蚊感染瘧原蟲(chóng)能力提高,有利于瘧疾的流行和擴(kuò)散。這將對(duì)如何控制瘧疾流行、媒介傳播提出了新的問(wèn)題,如何合理使用青蒿素類(lèi)殺紅內(nèi)期藥物提出了新的研究課題。
[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.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R392

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本文編號(hào)：1366130