TLRs激動劑對紅外期瘧原蟲發(fā)育的影響與機制研究
發(fā)布時間：2019-06-09 13:53
【摘要】： 瘧疾是蚊媒傳播的嚴(yán)重危害人類健康的熱帶傳染病,在全世界人群中具有很高的發(fā)病率和致死率。按蚊叮刺時將瘧原蟲子孢子注入宿主體內(nèi),隨后子孢子進(jìn)入血液循環(huán),并可在數(shù)分鐘后迅速侵入肝細(xì)胞。經(jīng)短暫的紅外期增殖發(fā)育,裂殖子再次進(jìn)入血液循環(huán)、侵入紅細(xì)胞導(dǎo)致瘧疾臨床發(fā)作。瘧原蟲在紅外期的增殖發(fā)育是瘧疾生活史中的重要階段,而阻斷蟲體在肝臟內(nèi)的增殖發(fā)育,即可防止瘧原蟲紅內(nèi)期感染。另外,紅外期休眠子的存在是導(dǎo)致間日瘧原蟲臨床病例復(fù)發(fā)的重要原因。因此,紅外期是制定干預(yù)瘧原蟲感染措施的理想時期,阻斷紅外期瘧原蟲生長發(fā)育能從源頭控制瘧原蟲的感染。然而,目前尚無有效的紅外期疫苗和預(yù)防藥物,迫切需要新的瘧疾防治手段和策略。 子孢子經(jīng)按蚊叮咬注入宿主后,必須且只有侵入肝細(xì)胞并在肝細(xì)胞內(nèi)進(jìn)行發(fā)育。鼠瘧在肝細(xì)胞內(nèi)43h后發(fā)育成熟,隨后肝細(xì)胞逐漸破裂并釋放出裂殖子。同樣,人瘧的紅外期周期約為1周~10多天,裂殖子進(jìn)入血液循環(huán)后發(fā)育成紅內(nèi)期瘧原蟲,將不會再返回肝臟。紅內(nèi)期瘧原蟲能夠反復(fù)感染紅細(xì)胞,宿主能夠產(chǎn)生適應(yīng)性免疫。然而,紅外期是瘧原蟲侵入宿主的早期,周期短暫,宿主不能及時產(chǎn)生適應(yīng)性免疫,主要依賴天然免疫抑制子孢子入侵肝細(xì)胞。在子孢子侵入過程中,大量的天然免疫細(xì)胞,如DCs、NK、NKT、γδT等細(xì)胞被激活,通過細(xì)胞毒效應(yīng)或分泌細(xì)胞因子IL-12、IFN-γ、TNF-α等抑制瘧原蟲子孢子感染肝細(xì)胞,并能夠影響CD4+和CD8+ T細(xì)胞對紅外期瘧原蟲的免疫應(yīng)答類型。因此,天然免疫對抑制紅外期瘧原蟲發(fā)育十分重要,探討宿主天然免疫抗紅外期瘧原蟲機制是制定新的瘧疾防治措施的前提。 TLRs(Toll-like receptors)受體是天然免疫細(xì)胞的重要模式識別分子,其介導(dǎo)的信號是天然免疫的重要組成部分。不同的TLRs能識別不同的病原體成分,如TLR2識別脂蛋白(Lipoprotein)、磷壁酸(Lipoteinchoic acid, LTA)和酵母聚糖(Zymosan),TLR3識別病毒dsRNA,TLR4識別脂多糖(LPS),TLR5識別鞭毛蛋白(Flagellin),TLR7/8識別小分子化合物如咪喹莫特(Imiquimod)和單鏈RNA,而TLR9特異性識別細(xì)菌DNA中的非甲基化CpG-ODN序列。TLRs表達(dá)于多種天然免疫細(xì)胞,如巨噬細(xì)胞,DC細(xì)胞,B細(xì)胞等。這些天然免疫細(xì)胞能在感染早期通過TLRs受體識別入侵的病原體而被活化,進(jìn)而激活NF-κB和轉(zhuǎn)錄因子AP-1,促進(jìn)IL-12、TNF-α、IL-1β和IL-6等炎癥因子的大量分泌,從而抑制病原體的增殖和擴散。研究證實,TLRs信號通路提前活化后能有效地抑制弓形蟲、細(xì)菌和病毒的感染。然而,目前還沒有資料顯示提前活化TLRs信號通路能否影響且有效抑制紅外期瘧原蟲的增殖與發(fā)育。 本研究通過約氏瘧原蟲-BABL/c小鼠模型,以肝臟蟲荷和原蟲血癥為檢測指標(biāo),對不同TLRs激動劑在紅外期瘧原蟲增殖發(fā)育中的作用進(jìn)行研究。其實驗內(nèi)容和結(jié)果主要包括以下四個方面: 一、構(gòu)建約氏瘧原蟲BY265株18S rRNA重組質(zhì)粒:通過對多種瘧原蟲18S rRNA基因的生物信息學(xué)分析,根據(jù)保守序列設(shè)計P.y. BY265株18S rRNA的特異性引物并進(jìn)行分子克隆。重組質(zhì)粒測序結(jié)果顯示該片段長度為839bp,經(jīng)Blast分析發(fā)現(xiàn)它與約氏瘧原蟲17XNL株相似性為98%。 二、構(gòu)建Real-time PCR紅外期瘧原蟲檢測平臺:在P.y. BY265株18S rRNA重組質(zhì)粒的基礎(chǔ)上,設(shè)計Real-time PCR特異性的引物和TaqMan探針,并與小鼠GAPDH特異性的Real-time PCR引物和TaqMan探針共同組成紅外期瘧原蟲Real-time PCR檢測平臺。以約氏瘧原蟲-BABL/c小鼠模型,通過對50、100、500和1000個唾液腺子孢子感染小鼠的肝臟蟲荷進(jìn)行定量分析,結(jié)果顯示Real-time PCR檢測平臺能夠檢測到最低為50個子孢子劑量感染的小鼠肝臟蟲荷。 三、以Real-time PCR為基礎(chǔ),系統(tǒng)的對不同TLRs激動劑在紅外期瘧原蟲增殖發(fā)育中的作用和影響進(jìn)行了研究:在單個TLRs激動劑尾靜脈注入小鼠24h后,將100個成熟的唾液腺子孢子再注入小鼠,部分小鼠在瘧原蟲感染后42h取出肝臟進(jìn)行Real-time PCR分析,部分小鼠在瘧原蟲感染后4~14天內(nèi)進(jìn)行原蟲血癥檢查。結(jié)果顯示,在該劑量下部分TLRs激動劑能夠顯著抑制紅外期瘧原蟲的發(fā)育,而部分TLRs激動劑對紅外期瘧原蟲的發(fā)育無顯著影響,反而對紅內(nèi)期的瘧原蟲發(fā)育具有一定的促進(jìn)作用: 1.在等量TLRs激動劑的作用下,TLR2、TLR3、TLR4和TLR9激動劑能夠顯著減少小鼠的肝臟蟲荷,其中以TLR9激動劑的作用最強,不僅能夠?qū)?00個唾液腺子孢子感染小鼠的肝臟蟲荷減少約90%,而且還能推遲紅內(nèi)期原蟲血癥的出現(xiàn)。當(dāng)增加瘧原蟲子孢子感染劑量后,肝臟蟲荷和原蟲血癥均有一定的增高,但與對照組相比,TLR9激動劑均能顯著減少肝臟蟲荷及延遲原蟲血癥發(fā)生。因此,活化TLR9信號通路誘導(dǎo)的免疫應(yīng)答在抑制紅外期瘧原蟲增殖發(fā)育中具有重要的作用。 2.與其它激動劑相同劑量的TLR5和TLR7激動劑對瘧原蟲感染小鼠的肝臟蟲荷變化無顯著影響。然而,原蟲血癥結(jié)果顯示,TLR5激動劑處理組小鼠的紅內(nèi)期原蟲血癥在感染8天后高于對照組,提示激活TLR5信號通路可能有利于紅內(nèi)期瘧原蟲的增殖發(fā)育。 四、探討TLR9激動劑和TLR5激動劑影響瘧原蟲發(fā)育的免疫機制: 1.TLR9激動劑與枯否細(xì)胞:枯否細(xì)胞(Kupffer cells,KC)是瘧原蟲子孢子侵入肝細(xì)胞的重要門戶。在氯化釓(GdCl3)特異性阻斷KC的吞噬功能后,TLR9激動劑抑制紅外期瘧原蟲發(fā)育的功能顯著下降,提示TLR9激動劑通過增強KC細(xì)胞吞噬功能抑制紅外期瘧原蟲的發(fā)育;然而,GdCl3無法完全消除TLR9激動劑的抑制作用,提示TLR 9激動劑還能激活其它的抗瘧原蟲免疫機制。 2.TLR9激動劑與肝臟細(xì)胞因子:細(xì)胞因子是重要的抗瘧原蟲效應(yīng)分子。本課題在進(jìn)一步研究中發(fā)現(xiàn),TLR9激動劑能夠上調(diào)肝臟內(nèi)促炎癥因子IFN-γ、IL-12、TNF-α基因的mRNA表達(dá),而下調(diào)抗炎癥因子IL-10和TGF-β基因的mRNA表達(dá),提示TLR9激動劑能夠誘導(dǎo)產(chǎn)生肝臟細(xì)胞因子抑制紅外期瘧原蟲的發(fā)育。 3.TLR5激動劑與紅內(nèi)期瘧原蟲:將TLR5重組真核表達(dá)質(zhì)粒轉(zhuǎn)染HEK293細(xì)胞,經(jīng)瘧原蟲感染的紅細(xì)胞及其裂解物分別刺激后,雙熒光素酶實驗結(jié)果顯示實驗組的熒光相對比值顯著增高,且完整的感染紅細(xì)胞刺激組顯著高于其裂解物刺激組,提示瘧原蟲感染的紅細(xì)胞能夠激活TLR5信號通路及感染的紅細(xì)胞膜上可能存在TLR5的配體。根據(jù)文獻(xiàn)推測,該配體可能通過活化TLR5信號通路激活調(diào)節(jié)性T細(xì)胞(Treg)從而降低機體對病原體的免疫應(yīng)答。 本課題通過建立有效的紅外期瘧原蟲定量檢測平臺,系統(tǒng)觀察提前24h激活TLRs信號通路對紅外期瘧原蟲發(fā)育的影響。TLR2、TLR3、TLR4和TLR9激動劑能夠顯著抑制紅外期瘧原蟲的發(fā)育,其中TLR9激動劑抑制作用最強,能夠顯著抑制瘧原蟲感染小鼠的肝臟蟲荷并推遲原蟲血癥的出現(xiàn)。深入研究發(fā)現(xiàn)TLR9激動劑通過增強KC細(xì)胞吞噬功能和誘導(dǎo)產(chǎn)生肝臟細(xì)胞因子等免疫效應(yīng)抑制紅外期瘧原蟲的發(fā)育;然而,相同劑量的TLR5和TLR7激動劑,對紅外期瘧原蟲的發(fā)育無顯著影響。但有意思的是,TLR5激動劑處理小鼠的原蟲血癥高于對照組,且在雙熒光素酶實驗中發(fā)現(xiàn)紅內(nèi)期瘧原蟲能夠激活TLR5信號通路,提示紅內(nèi)期瘧原蟲可能存在TLR5激動劑。這些結(jié)果對于我們認(rèn)識抗瘧原蟲紅外期天然免疫機制和深入探索宿主的天然免疫識別及瘧原蟲的免疫逃避的關(guān)系,并可為瘧疾免疫防治提供理論依據(jù)。
[Abstract]:Malaria is a tropical infectious disease that is a serious threat to human health by mosquito-borne diseases, and has a high morbidity and mortality in the worldwide population. The sporozoites of the plasmodium are injected into the host body when the mosquito is stabbed, and then the sporozoites enter the blood circulation and can rapidly enter the liver cells after a few minutes. After a brief infrared period of proliferation and development, the merozoites enter the blood circulation again, and the invading red blood cells cause the clinical attack of the malaria. The proliferation and development of plasmodium in the infrared period is an important stage in the life history of malaria, and the proliferation and development of the insect body in the liver can be blocked, and the infection of the plasmodium in the red inner phase can be prevented. In addition, the existence of the dormancy of the infrared period is an important cause of the recurrence of the clinical cases of Plasmodium vivax. Therefore, the infrared period is an ideal period for the development of the infection of Plasmodium vivax, which can block the infection of the plasmodium from the source by blocking the growth and development of the plasmodium. However, there are currently no effective infrared vaccines and preventive drugs, and there is an urgent need for new methods and strategies for malaria control. After the sporozoites are injected into the host according to the bite of the mosquito, it is necessary and only to invade the liver cells and enter the liver cells. The mouse malaria developed in the liver for 43 h, and then the liver cells were gradually broken and released. In the same way, the period of the infrared period of the human malaria is about 1 week to 10 days, and after the merozoites enter the blood circulation, the plasmodium is developed into the red-period plasmodium, and will not be returned again. Back to the liver. Plasmodium vivax can repeatedly infect the red blood cells, and the host can produce the adaptation. However, the infrared period is the early stage of the invasion of the parasite into the host, the period is short, the host can not produce the adaptive immunity in time, mainly relying on the natural immunosuppression subspore invasion In the process of subspore invasion, a large amount of natural immune cells, such as DCs, NK, NKT, and T-like cells, are activated to inhibit the infection of the sporozoites of the plasmodium by the cytotoxic effect or the secretion of the cytokine IL-12, IFN-1, TNF-1, and the like. The immune response of the CD4 + and CD8 + T cells to the plasmodium of the infrared period can be influenced by the liver cells. Therefore, the natural immunity is very important to the inhibition of the development of plasmodium in the infrared period, and the mechanism of the host natural immune anti-infrared period is to develop new malaria control measures The TLRs (Toll-like receptors) receptor is an important pattern recognition molecule of natural immune cells. The different TLRs can identify different pathogen components, such as TLR2, Lipoprotein, LTA and Zymosan, TLR3 recognizes the viral dsRNA, TLR4 recognizes lipopolysaccharide (LPS), TLR5 recognizes flagellin (Fla). gellin), TLR7/8 recognizes a small molecule compound such as Imiquimod and single-stranded RNA, while TLR9 specifically recognizes the non-methylated Cp in the bacterial DNA G-ODN sequence. TLRs are expressed in a variety of natural immune cells, such as macrophages, DCs, The natural immune cells can be activated by identifying the invading pathogens in the early stage of infection by the TLRs receptor, so as to activate the NF-B and the transcription factor AP-1 to promote a large amount of secretion of the inflammatory factors such as IL-12, TNF-1, IL-1, and IL-6, thereby inhibiting the pathogen. It is proved that the TLRs can effectively inhibit Toxoplasma gondii and fine after the early activation of the signal pathway of the TLRs. However, there is no information on whether the early activation of the TLRs signal pathway can affect and effectively inhibit the infrared malarial. The effects of different TLRs on the proliferation and development of the worm were studied by using the model of Plasmodium yoelii-BABL/ c. A study of the role of the development of colonisation. The results mainly include the following four aspects:1. Construction of the 18S rRNA recombinant plasmid of the BY265 strain of the Plasmodium yoelii. The 18S rR of the P. y. BY265 strain 18S rR is designed according to the conserved sequence by the bioinformatics analysis of the 18S rRNA gene of the plasmodium. The specific primers of NA and the molecular cloning were carried out. The results of the recombinant plasmid sequencing showed that the length of the fragment was 839 bp, and it was found to be in the form of some malaria by the Blast analysis. A real-time PCR-specific primer and a TaqMan probe were designed on the basis of the 18S rRNA recombinant plasmid of P. y. BY265, and the real-time PCR primer and the TaqMan probe specific to the mouse GAPDH were combined to form the infrared phase. Plasmodium vivax-BABL/ c mouse model, a quantitative analysis of the liver worm of mice infected with 50,100,500 and 1000 salivary glands was carried out, and the results showed that the real-time PCR detection platform was able to detect the most The effects and effects of different TLRs agonists on the proliferation and development of Plasmodium vivax were studied on the basis of Real-time PCR. After 4 h,100 mature salivary gland subspores were re-injected into the mice, and some of the mice were taken out of the liver for real-time PCR analysis at 42 h after the infection of the plasmodium. The results show that the partial TLRs agonist can significantly inhibit the development of the plasmodium in the infrared period, while the partial TLRs agonist has no significant effect on the development of the plasmodium in the infrared period. In response, the effects of TLR2, TLR3, TLR4 and TLR9 agonist on the development of the plasmodium in the red inner stage can significantly reduce the liver insect-loading of the mice, and the effect of the TLR9 agonist is the strongest, not only can the TLR2, TLR3, TLR4 and TLR9 agonists Enough to infect the 100 salivary gland subspores to the mice. The decrease of the liver worm was reduced by about 90%, and the occurrence of the endotoxemia in the red endothelia could be delayed. After the infection of the sporozoites of the plasmodium, the liver and the protozoan were increased, but compared with the control group. The TLR9 agonist can significantly reduce the occurrence of hepatic and delayed protozoan. Therefore, the activation of TLR9 signaling pathway the immune response of the derivative plays an important role in the inhibition of the proliferation and development of the Plasmodium vivax.2. The same dose of T as other agonists The LR5 and TLR7 agonists have no significant effect on the changes in the liver of the mice infected with plasmodium. However, the results of the protozoa show that the red endotoxemia of the TLR5 agonist treatment group mice is higher than in the case of 8 days of infection In group, it is suggested that the activation of TLR5 signal pathway may be beneficial to the proliferation and development of Plasmodium vivax. 4. To study the immune mechanism of TLR9 agonists and TLR5 agonists to the development of plasmodium:1. TLR9 agonists and dead cells : Kupffer cells (KC) is an important portal for the invasion of the sporozoites of the plasmodium. After the specific blocking of the phagocytosis of the KC, the TLR9 agonist inhibits the development of the Plasmodium vivax. It is suggested that the TLR9 agonist can inhibit the development of the infrared stage plasmodium by enhancing the phagocytosis of the KC cells; however, the GdCl3 can not be completely The inhibition of TLR9 agonists is eliminated, suggesting that the TLR 9 agonist can also activate other antimalarial precursors in a further study, TLR9 agonists are able to upregulate that inflammatory factor IFN-1, IL-12 in the liver, The mRNA expression of the TNF-1 gene and the down-regulation of the anti-inflammatory factors IL-10 and TGF- 3. TLR5 agonists and plasmodium vivax: the recombinant eukaryotic expression plasmid of TLR5 was transfected into HEK293 cells. After stimulation of the red blood cells and the lysates of the plasmodium, the results of the two-luciferase experiment show that the relative ratio of the fluorescence in the experimental group is significantly higher, and the complete infection of the red blood cell stimulation group is significantly higher than the lysate thereof. In the stimulation group, the red blood cells infected with the plasmodium can activate the TLR5 signaling pathway and the potential TLR5 ligand on the infected red cell membrane. The ligand may activate regulatory T cells (Treg) by activating the TLR5 signaling pathway to reduce the immune response of the body to the pathogen. The effects of the activation of TLRs on the development of Plasmodium vivax were observed by establishing an effective quantitative detection platform for Plasmodium vivax. TLR2, TLR3, TLR4 and TLR9 agonists can significantly inhibit the infrared period. The development of the plasmodium, in which the inhibitory effect of the TLR9 agonist is the strongest, can significantly inhibit the liver of the mice infected with the plasmodium, and delay the occurrence of the protozoan. In-depth study of the discovery of TLR9 agonists by enhancing the phagocytosis of the KC and the induction of the production of the liver cytokines, etc. The effects of phytophthora blight on the development of plasmodium in the infrared period were inhibited; however, the same dose of TLR5 and TLR7 agonists had no significant effect on the development of Plasmodium vivax, but it was interesting that the TLR5 agonist treated mice with a higher protozoan effect than that of the control group. In that double-luciferase experiment, the TLR5 signal pathway can be activated by the plasmodium vivax, suggesting that a TLR5 agonist may be present in the red endoplasmodium.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R392
本文編號：2495602
[Abstract]:Malaria is a tropical infectious disease that is a serious threat to human health by mosquito-borne diseases, and has a high morbidity and mortality in the worldwide population. The sporozoites of the plasmodium are injected into the host body when the mosquito is stabbed, and then the sporozoites enter the blood circulation and can rapidly enter the liver cells after a few minutes. After a brief infrared period of proliferation and development, the merozoites enter the blood circulation again, and the invading red blood cells cause the clinical attack of the malaria. The proliferation and development of plasmodium in the infrared period is an important stage in the life history of malaria, and the proliferation and development of the insect body in the liver can be blocked, and the infection of the plasmodium in the red inner phase can be prevented. In addition, the existence of the dormancy of the infrared period is an important cause of the recurrence of the clinical cases of Plasmodium vivax. Therefore, the infrared period is an ideal period for the development of the infection of Plasmodium vivax, which can block the infection of the plasmodium from the source by blocking the growth and development of the plasmodium. However, there are currently no effective infrared vaccines and preventive drugs, and there is an urgent need for new methods and strategies for malaria control. After the sporozoites are injected into the host according to the bite of the mosquito, it is necessary and only to invade the liver cells and enter the liver cells. The mouse malaria developed in the liver for 43 h, and then the liver cells were gradually broken and released. In the same way, the period of the infrared period of the human malaria is about 1 week to 10 days, and after the merozoites enter the blood circulation, the plasmodium is developed into the red-period plasmodium, and will not be returned again. Back to the liver. Plasmodium vivax can repeatedly infect the red blood cells, and the host can produce the adaptation. However, the infrared period is the early stage of the invasion of the parasite into the host, the period is short, the host can not produce the adaptive immunity in time, mainly relying on the natural immunosuppression subspore invasion In the process of subspore invasion, a large amount of natural immune cells, such as DCs, NK, NKT, and T-like cells, are activated to inhibit the infection of the sporozoites of the plasmodium by the cytotoxic effect or the secretion of the cytokine IL-12, IFN-1, TNF-1, and the like. The immune response of the CD4 + and CD8 + T cells to the plasmodium of the infrared period can be influenced by the liver cells. Therefore, the natural immunity is very important to the inhibition of the development of plasmodium in the infrared period, and the mechanism of the host natural immune anti-infrared period is to develop new malaria control measures The TLRs (Toll-like receptors) receptor is an important pattern recognition molecule of natural immune cells. The different TLRs can identify different pathogen components, such as TLR2, Lipoprotein, LTA and Zymosan, TLR3 recognizes the viral dsRNA, TLR4 recognizes lipopolysaccharide (LPS), TLR5 recognizes flagellin (Fla). gellin), TLR7/8 recognizes a small molecule compound such as Imiquimod and single-stranded RNA, while TLR9 specifically recognizes the non-methylated Cp in the bacterial DNA G-ODN sequence. TLRs are expressed in a variety of natural immune cells, such as macrophages, DCs, The natural immune cells can be activated by identifying the invading pathogens in the early stage of infection by the TLRs receptor, so as to activate the NF-B and the transcription factor AP-1 to promote a large amount of secretion of the inflammatory factors such as IL-12, TNF-1, IL-1, and IL-6, thereby inhibiting the pathogen. It is proved that the TLRs can effectively inhibit Toxoplasma gondii and fine after the early activation of the signal pathway of the TLRs. However, there is no information on whether the early activation of the TLRs signal pathway can affect and effectively inhibit the infrared malarial. The effects of different TLRs on the proliferation and development of the worm were studied by using the model of Plasmodium yoelii-BABL/ c. A study of the role of the development of colonisation. The results mainly include the following four aspects:1. Construction of the 18S rRNA recombinant plasmid of the BY265 strain of the Plasmodium yoelii. The 18S rR of the P. y. BY265 strain 18S rR is designed according to the conserved sequence by the bioinformatics analysis of the 18S rRNA gene of the plasmodium. The specific primers of NA and the molecular cloning were carried out. The results of the recombinant plasmid sequencing showed that the length of the fragment was 839 bp, and it was found to be in the form of some malaria by the Blast analysis. A real-time PCR-specific primer and a TaqMan probe were designed on the basis of the 18S rRNA recombinant plasmid of P. y. BY265, and the real-time PCR primer and the TaqMan probe specific to the mouse GAPDH were combined to form the infrared phase. Plasmodium vivax-BABL/ c mouse model, a quantitative analysis of the liver worm of mice infected with 50,100,500 and 1000 salivary glands was carried out, and the results showed that the real-time PCR detection platform was able to detect the most The effects and effects of different TLRs agonists on the proliferation and development of Plasmodium vivax were studied on the basis of Real-time PCR. After 4 h,100 mature salivary gland subspores were re-injected into the mice, and some of the mice were taken out of the liver for real-time PCR analysis at 42 h after the infection of the plasmodium. The results show that the partial TLRs agonist can significantly inhibit the development of the plasmodium in the infrared period, while the partial TLRs agonist has no significant effect on the development of the plasmodium in the infrared period. In response, the effects of TLR2, TLR3, TLR4 and TLR9 agonist on the development of the plasmodium in the red inner stage can significantly reduce the liver insect-loading of the mice, and the effect of the TLR9 agonist is the strongest, not only can the TLR2, TLR3, TLR4 and TLR9 agonists Enough to infect the 100 salivary gland subspores to the mice. The decrease of the liver worm was reduced by about 90%, and the occurrence of the endotoxemia in the red endothelia could be delayed. After the infection of the sporozoites of the plasmodium, the liver and the protozoan were increased, but compared with the control group. The TLR9 agonist can significantly reduce the occurrence of hepatic and delayed protozoan. Therefore, the activation of TLR9 signaling pathway the immune response of the derivative plays an important role in the inhibition of the proliferation and development of the Plasmodium vivax.2. The same dose of T as other agonists The LR5 and TLR7 agonists have no significant effect on the changes in the liver of the mice infected with plasmodium. However, the results of the protozoa show that the red endotoxemia of the TLR5 agonist treatment group mice is higher than in the case of 8 days of infection In group, it is suggested that the activation of TLR5 signal pathway may be beneficial to the proliferation and development of Plasmodium vivax. 4. To study the immune mechanism of TLR9 agonists and TLR5 agonists to the development of plasmodium:1. TLR9 agonists and dead cells : Kupffer cells (KC) is an important portal for the invasion of the sporozoites of the plasmodium. After the specific blocking of the phagocytosis of the KC, the TLR9 agonist inhibits the development of the Plasmodium vivax. It is suggested that the TLR9 agonist can inhibit the development of the infrared stage plasmodium by enhancing the phagocytosis of the KC cells; however, the GdCl3 can not be completely The inhibition of TLR9 agonists is eliminated, suggesting that the TLR 9 agonist can also activate other antimalarial precursors in a further study, TLR9 agonists are able to upregulate that inflammatory factor IFN-1, IL-12 in the liver, The mRNA expression of the TNF-1 gene and the down-regulation of the anti-inflammatory factors IL-10 and TGF- 3. TLR5 agonists and plasmodium vivax: the recombinant eukaryotic expression plasmid of TLR5 was transfected into HEK293 cells. After stimulation of the red blood cells and the lysates of the plasmodium, the results of the two-luciferase experiment show that the relative ratio of the fluorescence in the experimental group is significantly higher, and the complete infection of the red blood cell stimulation group is significantly higher than the lysate thereof. In the stimulation group, the red blood cells infected with the plasmodium can activate the TLR5 signaling pathway and the potential TLR5 ligand on the infected red cell membrane. The ligand may activate regulatory T cells (Treg) by activating the TLR5 signaling pathway to reduce the immune response of the body to the pathogen. The effects of the activation of TLRs on the development of Plasmodium vivax were observed by establishing an effective quantitative detection platform for Plasmodium vivax. TLR2, TLR3, TLR4 and TLR9 agonists can significantly inhibit the infrared period. The development of the plasmodium, in which the inhibitory effect of the TLR9 agonist is the strongest, can significantly inhibit the liver of the mice infected with the plasmodium, and delay the occurrence of the protozoan. In-depth study of the discovery of TLR9 agonists by enhancing the phagocytosis of the KC and the induction of the production of the liver cytokines, etc. The effects of phytophthora blight on the development of plasmodium in the infrared period were inhibited; however, the same dose of TLR5 and TLR7 agonists had no significant effect on the development of Plasmodium vivax, but it was interesting that the TLR5 agonist treated mice with a higher protozoan effect than that of the control group. In that double-luciferase experiment, the TLR5 signal pathway can be activated by the plasmodium vivax, suggesting that a TLR5 agonist may be present in the red endoplasmodium.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R392
【共引文獻(xiàn)】
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1 荊科;孫梅;;腸三葉因子對內(nèi)毒素血癥幼鼠腸組織Toll樣受體2和4表達(dá)的影響[J];中國當(dāng)代兒科雜志;2011年12期
2 楊坤;汪霞;魏聰;黃媛;付學(xué)東;趙東赤;;呼吸道合胞病毒激活RIG-Ⅰ/TLR3信號通路及對SOCS1/3 mRNA表達(dá)的影響[J];武漢大學(xué)學(xué)報(醫(yī)學(xué)版);2010年05期
3 張曉東;方敬愛;孫艷艷;劉文媛;;TLR4與腎小球疾病[J];中國中西醫(yī)結(jié)合腎病雜志;2009年01期
4 ;Toll-like receptors,a double-edged sword in immunity to malaria[J];Journal of Medical Colleges of PLA;2009年02期
5 吳燕燕;王易;;Toll樣受體信號通路中MyD88的研究進(jìn)展[J];免疫學(xué)雜志;2012年03期
6 李文新;王嘉軍;;Toll樣受體直接調(diào)節(jié)Treg細(xì)胞抑制功能的研究進(jìn)展[J];生命科學(xué);2012年01期
7 賈玉臣;陳慶森;;生物活性肽對腸黏膜免疫調(diào)節(jié)作用的研究進(jìn)展[J];食品科學(xué);2009年21期
8 張滟;許珉;張增鐵;;中耳膽脂瘤上皮中TLR2表達(dá)的研究[J];陜西醫(yī)學(xué)雜志;2008年08期
9 劉興華;彭文輝;李海玲;張戟;聞國富;徐亞偉;;高糖誘導(dǎo)人THP1細(xì)胞Toll樣受體4信號通路變化及匹伐他汀的拮抗作用[J];同濟大學(xué)學(xué)報(醫(yī)學(xué)版);2011年04期
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相關(guān)博士學(xué)位論文 前10條
1 鄧緒芳;p53在日本腦炎病毒復(fù)制及其致病性中的作用[D];中國農(nóng)業(yè)科學(xué)院;2011年
2 謝立蘭;偽狂犬病毒和豬傳染性胃腸炎病毒誘導(dǎo)β干擾素產(chǎn)生的分子機制研究[D];華中農(nóng)業(yè)大學(xué);2011年
3 閔安杰;RNA干擾沉默TREM-1對脆弱類桿菌脂多糖炎癥反應(yīng)的抑制作用及其機制研究[D];中南大學(xué);2007年
4 王晶;Toll樣受體3、9在皮膚中的作用機制和信號轉(zhuǎn)導(dǎo)通路研究及藥物的干預(yù)[D];第二軍醫(yī)大學(xué);2007年
5 丁彥春;模式識別受體在血管內(nèi)膜增生及血管平滑肌細(xì)胞激活中的作用[D];大連醫(yī)科大學(xué);2008年
6 鄧潔捷;納米材料對小鼠肺毒性的研究和甲型H1N1流感病毒在人肺上皮細(xì)胞及雪貂中的致病性研究[D];北京協(xié)和醫(yī)學(xué)院;2012年
7 湯小燕;TLR4信號通路參與人結(jié)腸癌細(xì)胞免疫逃逸機制的研究[D];武漢大學(xué);2009年
8 趙一萍;蒙古馬免疫相關(guān)基因表達(dá)研究及脾臟表達(dá)譜分析[D];內(nèi)蒙古農(nóng)業(yè)大學(xué);2013年
9 朱紫祥;p53在I型干擾素介導(dǎo)的先天性抗甲型流感病毒中的作用研究[D];中國農(nóng)業(yè)科學(xué)院;2013年
10 朱亞軍;Toll樣受體家族與高脂血癥和胰島素抵抗的相關(guān)性研究[D];河北醫(yī)科大學(xué);2014年
相關(guān)碩士學(xué)位論文 前7條
1 魏巴金;Toll樣受體家族基因多態(tài)性與肝移植后乙肝復(fù)發(fā)的關(guān)系[D];浙江大學(xué);2011年
2 吳紅;Toll樣受體7在HBV感染中作用及相關(guān)機制的研究[D];廣州醫(yī)學(xué)院;2010年
3 黃禮彬;角膜基質(zhì)細(xì)胞Toll樣受體4對茄病鐮刀菌炎性反應(yīng)的實驗研究[D];福建醫(yī)科大學(xué);2009年
4 任文智;云芝糖肽（PSP）對小鼠巨噬細(xì)胞的雙向免疫調(diào)節(jié)及NF-κB信號通路的調(diào)控研究[D];上海師范大學(xué);2010年
5 劉殿波;人類新型冠狀病毒NL63木瓜樣蛋白酶生物學(xué)功能研究[D];內(nèi)蒙古農(nóng)業(yè)大學(xué);2010年
6 陳銘;CpG-c41對TLRs信號通路的交互影響及其抗類風(fēng)濕關(guān)節(jié)炎的初步研究[D];第三軍醫(yī)大學(xué);2013年
7 晏星;豬GBP1、GBP2基因的克隆及其對PRRSV和PRV增殖的影響[D];華中農(nóng)業(yè)大學(xué);2014年
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