約氏瘧原蟲感染小鼠巨噬細胞活化和效應特點的研究
發(fā)布時間：2018-08-29 19:54
【摘要】： 前言 瘧疾是全球關注、高發(fā)的感染性疾病。Nature最新報道顯示，截止2002年，世界感染瘧疾的患者人數(shù)達5.15億，全球受威脅人口達22億。為此，WHO將艾滋病、結(jié)核和瘧疾列為三類優(yōu)先重點防治的感染性疾病。目前，瘧疾疫苗和抗瘧新藥的研制與開發(fā)是有效控制瘧疾發(fā)生和發(fā)展的重要戰(zhàn)略，其必需的前提基礎有賴于對宿主保護性免疫應答機制的進一步闡明。 單核-巨噬細胞在不同方面明顯影響著瘧原蟲的感染過程，它既可作為非特異免疫系統(tǒng)細胞發(fā)揮非特異性吞噬、殺傷作用，也可參與介導特異性免疫應答，發(fā)揮抗原提呈和免疫調(diào)節(jié)的重要作用。大量研究表明，紅內(nèi)期保護性免疫應答的鮮明特點是Th1和Th2型免疫應答的有序活化，由CD_4~+Yh細胞和抗體共同參與。感染急性期以IFN-γ為主導的Th1型炎癥性細胞因子活化巨噬細胞等免疫效應細胞，遏制瘧原蟲在紅內(nèi)期的爆發(fā)性增殖，有效控制原蟲血癥的迅速上升：隨后應答向Th2型過渡，最終以抗體介導的免疫效應徹底清除瘧原蟲。我們的前期研究已證實，活化的單核-巨噬細胞在約氏瘧原蟲感染早期可能是遏制蟲體血癥水平的關鍵因素之一。因此，有必要對巨噬細胞在整個感染過程中的活化規(guī)律和效應特點進行更深一步探討和闡明。 本研究選用P．y 17XL抵抗型DBA／2小鼠為研究對象，首次全程動態(tài)觀察巨噬細胞表面膜分子、效應分子NO含量及其吞噬功能的變化，以期進一步闡明在瘧疾感染中巨噬細胞的活化規(guī)律和效應特點，旨在為有效瘧疾疫苗和抗瘧新藥的研制、開發(fā)提供新的理論和實驗依據(jù)。 實驗材料與方法 本研究選用P．y 17XL抵抗型DBA／2小鼠為研究對象。Giemsa薄血膜染色，光學顯微鏡計數(shù)紅細胞感染率；流式細胞技術(shù)動態(tài)檢測DBA／2小鼠在P．y 17XL感染過程中，脾巨噬細胞數(shù)量，表面膜分子CD36、CD64和MHCⅡ的表達水平；通過Griess反應，測定脾巨噬細胞分泌NO_2~-的含量；顯微鏡檢巨噬細胞的吞噬功能。數(shù)據(jù)以均值士標準差((?)±s)表示，數(shù)據(jù)經(jīng)t檢驗，P＜0.05為差異顯著。 實驗結(jié)果 DBA／2小鼠在P．y 17XL攻擊后原蟲血癥緩慢上升，于感染后第7d達到峰值后逐漸降低，在感染后約第15d小鼠自愈，瘧原蟲感染的紅細胞被全部清除；于急性感染早期(3d-5d)，脾巨噬細胞NO分泌水平出現(xiàn)有意義的升高，繼而巨噬細胞吞噬能力于感染后第5d開始增強，至感染后第10d吞噬率高達95％，隨之持續(xù)維持于高水平；脾巨噬細胞表面MHCⅡ類分子和CD36于感染后第3d開始升高，至感染后第10d分別達到峰值；CD64表達水平于感染后第5d開始升高，隨后持續(xù)維持于高水平；并且，巨噬細胞數(shù)量也于感染后第3d開始明顯增加，至第10d達到峰值。 討論 近年來，許多研究表明天然免疫和獲得性免疫應答參與控制紅內(nèi)期瘧原蟲感染，減輕原蟲負荷，延緩發(fā)展為重癥瘧疾的感染進程。巨噬細胞具有顯著的非特異性吞噬和殺傷能力，經(jīng)其有效的刺激因子IFN-γ活化后其功能可進一步增強。以往報道顯示，體液循環(huán)的單核細胞和組織中(肝、脾)的巨噬細胞能夠通過清除受瘧原蟲感染的紅細胞(PRBC)參與控制瘧原蟲感染。我們的前期研究也已證實，抵抗型DBA／2小鼠CD_4~+Th1細胞于感染早期可產(chǎn)生高水平的IFN-γ，活化的巨噬細胞具有重要的紅內(nèi)期瘧原蟲增殖抑制效應。在此，本研究利用P．y 17XL抵抗型DBA／2小鼠模型，，系統(tǒng)地觀察了巨噬細胞在P．y 17XL感染過程中的活化規(guī)律和效應特點。實驗結(jié)果顯示，在P．y 17XL感染過程中，巨噬細胞于感染早期迅速活化，以分泌效應分子NO抑制原蟲血癥及非特異性吞噬方式殺傷瘧原蟲。由此提示， 巨噬細胞是DBA／2小鼠產(chǎn)生抗瘧保護性免疫應答的重要效應細胞。 以往研究顯示，作為專職抗原提呈細胞，巨噬細胞可通過表達MHC分子和共刺激分子提呈抗原給T細胞，為T細胞活化提供必要的活化信號并控制T細胞的分化方向從而影響瘧疾的感染結(jié)局。MHCⅡ類分子是巨噬細胞活化的重要標志。MHCⅡ敲除鼠研究顯示，非致死型約氏瘧原蟲和夏氏瘧原蟲感染紅內(nèi)期的保護性免疫完全依賴于MHCⅡ類分子。BALB／c小鼠感染非致死型約氏瘧原蟲后，巨噬細胞表達MHCⅡ和CD80的水平明顯增加，從而促進T細胞活化和IFN-γ的分泌。本研究進一步發(fā)現(xiàn)，P．y 17XL感染早期巨噬細胞MHCⅡ類分子表達水平明顯增強，同時伴隨巨噬細胞數(shù)量的顯著增加。提示巨噬細胞在IFN-γ刺激作用下發(fā)生了有效活化，活化的單核．巨噬細胞參與DBA／2小鼠Th1型免疫應答的有效建立。 已證實，巨噬細胞是非特異性免疫效應分子NO的重要來源，NO氧化后形成的過氧化氮是巨噬細胞發(fā)揮殺傷毒性的主要效應分子。體外實驗顯示，NO在紅內(nèi)期發(fā)揮細胞毒性和原蟲生長抑制作用。盡管NO在抗紅內(nèi)期瘧原蟲免疫機制中的作用尚存爭議，但是我們前期研究已經(jīng)發(fā)現(xiàn)，約氏瘧原蟲感染早期隨著DBA／2小鼠NO合成的增加，紅細胞感染率不斷上升的趨勢受到遏制，并可見網(wǎng)織紅細胞的表面出現(xiàn)許多裂殖子附著現(xiàn)象。推測可能是因為感染早期NO合成水平的提高使大量裂殖子對紅細胞侵襲能力發(fā)生障礙。本研究結(jié)果顯示，盡管DBA／2小鼠于感染后前4d巨噬細胞尚未表現(xiàn)吞噬作用，但小鼠脾細胞培養(yǎng)上清中NO水平于感染后第3d即出現(xiàn)顯著升高。與此同時，原蟲血癥水平呈現(xiàn)緩慢上升趨勢。由此提示，在瘧疾感染早期巨噬細胞可通過合成分泌NO發(fā)揮原蟲抑制作用。 巨噬細胞的非特異性吞噬包括非調(diào)理性吞噬和調(diào)理性吞噬兩種機制。巨噬細胞的非調(diào)理性吞噬通過識別病原體表面模式識別受體包括清道夫受體和甘露糖受體完成。CD36是清道夫受體家族B型受體，目前認為CD36在介導巨噬細胞非調(diào)理性吞噬功能中發(fā)揮重要作用。一些研究顯示，CD36與瘧原蟲感染紅細胞的結(jié)合影響瘧疾發(fā)病的嚴重程度。CD36表達缺陷與重癥瘧疾(尤其是腦瘧)關系密切。上調(diào)巨噬細胞表面CD36分子后，人及C57BL／6、BALB／c小鼠的巨噬細胞對瘧原蟲感染紅細胞的吞噬活性明顯增強；而應用抗CD36單克隆抗體后其吞噬能力下降。通過受體阻斷和CD36基因敲除的研究發(fā)現(xiàn)：在體外缺乏調(diào)理作用的情況下，人和小鼠的巨噬細胞均能吞噬瘧原蟲環(huán)狀體期感染的紅細胞(RPEs)，此作用依賴于巨噬細胞表面的CD36分子，而非其它表面受體(ICAM-1，αvβ3，或PECAM-1或模式識別受體如甘露糖受體或CD14)；此外，在單核巨噬細胞對惡性瘧原蟲配子體的清除中CD36亦發(fā)揮關鍵的天然免疫防御作用。本研究通過全程動態(tài)觀察發(fā)現(xiàn)，在P．y 17XL抵抗型DBA／2小鼠自然感染過程中，巨噬細胞表面CD36分子從感染后第3d表達水平開始明顯升高，隨感染進程至第10d達到峰值，相應巨噬細胞吞噬能力明顯增強。由此提示，瘧疾感染早期巨噬細胞可能通過CD36發(fā)揮非調(diào)理性吞噬作用。 瘧疾紅內(nèi)期保護性免疫應答經(jīng)過急性期感染階段后，最終要由特異性抗體介導的免疫應答清除瘧原蟲�？贵w介導的調(diào)理性吞噬可能是瘧原蟲感染紅內(nèi)期獲得性免疫應答重要機制之一。利用Carrageenan(巨噬細胞抑制劑)研究發(fā)現(xiàn)，抗體依賴的FcR介導的調(diào)理性吞噬殺傷是清除P．berghei XAT紅內(nèi)期感染的關鍵，提示脾巨噬細胞的吞噬活性對宿主抵抗瘧疾感染十分必要。FcRγ-／-(γ鏈敲除)小鼠由于缺乏抗體依賴性吞噬和抗體依賴的細胞毒作用(ADCC)，對P．berghei XAT易感性明顯增強并最終死亡。Mota等證實，CBA小鼠外周血巨噬細胞能夠?qū)ο氖席懺x(P．chabaudi chabaudi)感染紅細胞發(fā)揮抗體介導的調(diào)理性吞噬作用，且具有蟲株特異性。我們的前期研究顯示，DBA／2小鼠在致死型約氏瘧原蟲感染后第10d瘧原蟲特異性IgG抗體水平升高。本研究檢測到巨噬細胞表面的FcγRⅠ(CD64)從感染后第5d表達水平開始升高，隨后逐漸上升并持續(xù)維持較高水平。與此同時，巨噬細胞吞噬率在感染第8-15d處于高水平。由此提示，在約氏瘧原蟲感染后期，伴隨特異性抗體的產(chǎn)生，巨噬細胞可能通過CD64發(fā)揮調(diào)理性吞噬作用。 綜上，本研究通過全程、動態(tài)、系統(tǒng)的觀察抵抗型DBA／2小鼠的巨噬細胞在P．y 17XL自然感染過程中的活化及效應特點，進一步揭示了巨噬細胞無論在抗瘧原蟲感染的天然免疫和獲得性免疫中均發(fā)揮重要作用。巨噬細胞活化和效應特點及其相關機制的進一步闡明，必將為有效的瘧疾疫苗和抗瘧新藥的研制開發(fā)提供必要的理論和實驗依據(jù)。 結(jié)論 在致死型約氏瘧原蟲感染過程中，巨噬細胞無論在天然免疫和獲得性免疫中均發(fā)揮重要的作用。 1．DBA／2小鼠脾巨噬細胞的數(shù)量呈現(xiàn)逐步升高的動態(tài)變化趨勢； 2．通過MHCⅡ類分子表達，提示巨噬細胞在IFN-γ刺激作用下發(fā)生了有效活化； 3．巨噬細胞表達的清道夫受體CD36在感染早期顯著增加，介導非調(diào)理性吞噬殺傷作用； 4．作為巨噬細胞表達的FcγRI(CD64)在感染后期的顯著增加，可能參與抗體介導的調(diào)理性吞噬殺傷作用； 5．巨噬細胞分泌NO的水平于感染早期出現(xiàn)有意義的升高，由此抑制瘧原蟲的爆發(fā)性增殖。
[Abstract]:Preface
Malaria is a global concern and a high incidence of infectious diseases. According to the latest report from Nature, the number of people infected with malaria in the world reached 515 million by 2002, and the number of people threatened worldwide reached 2.2 billion. It is an important strategy to effectively control the occurrence and development of malaria, and its necessary prerequisite depends on the further elucidation of the host protective immune response mechanism.
Monocyte-macrophage plays a significant role in the infection process of Plasmodium in different aspects. It can not only act as a non-specific immune system cell to perform non-specific phagocytosis and kill, but also participate in the mediation of specific immune response and play an important role in antigen presentation and immune regulation. It is characterized by the orderly activation of Th1 and Th2 immune responses, with the participation of CD_4~+Yh cells and antibodies. In the acute phase of infection, IFN-gamma-dominated Th1 inflammatory cytokines activate immune effector cells such as macrophages, inhibit the explosive proliferation of Plasmodium in the intraerythrocytic phase, effectively control the rapid rise of protozoemia: the subsequent response to Th Our previous studies have shown that activated monocytes and macrophages may be one of the key factors to control the level of parasitemia in the early stage of Plasmodium yoelii infection. Further explore and clarify.
In this study, P.y 17XL-resistant DBA/2 mice were selected as the research objects. The changes of membrane molecules, NO content and phagocytic function of macrophages were observed for the first time. The aim of this study was to further clarify the activation rules and effect characteristics of macrophages in malaria infection, and to develop effective malaria vaccines and new antimalarial drugs. Development provides new theoretical and experimental basis.
Experimental materials and methods
In this study, P.y 17XL-resistant DBA/2 mice were selected as the research objects.Giemsa thin blood membrane staining and light microscopy were used to count the infection rate of red blood cells.Flow cytometry was used to detect the number of splenic macrophages and the expression levels of surface membrane molecules CD36, CD64 and MHC II during P.y 17XL infection in DBA/2 mice. The content of NO_2~- secreted by splenic macrophages and the phagocytic function of macrophages were examined by microscope.
experimental result
In DBA/2 mice, the level of protozomia increased slowly after P.y 17XL attack, decreased gradually after 7 days of infection, and all the erythrocytes infected by P.falciparum were cleared after 15 days of infection. In the early stage of acute infection (3-5 days), the level of NO secreted by splenic macrophages increased significantly, and then the phagocytic capacity of macrophages increased. The phagocytosis rate was as high as 95% on the 5th day after infection, and then remained at a high level on the 10th day after infection; the MHC class II molecule and CD36 on the surface of splenic macrophages began to increase on the 3rd day after infection and reached their peak value on the 10th day after infection; the expression of CD64 began to increase on the 5th day after infection, and then remained at a high level. The number of macrophages also increased significantly at 3D after infection and reached the peak at 10d.
discuss
In recent years, many studies have shown that innate and acquired immune responses participate in the control of erythrocytic malaria infection, reduce the burden of parasites, and delay the development of severe malaria infection. Previous studies have shown that monocytes circulating in body fluid and macrophages in tissues (liver, spleen) can participate in the control of malaria infection by eliminating the erythrocytes (PRBC) infected by Plasmodium. Our previous studies have also confirmed that CD_4~+Th1 cells of resistant DBA/2 mice can produce high levels of IFN-gamma in the early stage of infection, and activated macrophages possess high levels of IFN-gamma. In this study, the activation of macrophages during P.y 17XL infection was systematically observed in a P.y 17XL resistant DBA/2 mouse model. The results showed that macrophages were activated rapidly in the early stage of P.y 17XL infection in order to produce secretory effects. The molecule NO inhibits protozoa and non-specific phagocytosis to kill Plasmodium.
Macrophages are important effector cells in DBA / 2 mice producing antimalarial protective immune responses.
Previous studies have shown that as full-time antigen presenting cells, macrophages can present antigens to T cells by expressing MHC molecules and costimulatory molecules, providing necessary activation signals for T cell activation and controlling the direction of T cell differentiation, thereby affecting the outcome of malaria infection. MHC class II molecules are important markers of macrophage activation. The protective immunity of non-lethal Plasmodium yoelii and Plasmodium charantii during intraerythrocytic infection was completely dependent on MHC class II molecules. The expression of MHC class II and CD80 in macrophages of BALB/c mice infected with non-lethal Plasmodium yoelii increased significantly, thus promoting the activation of T cells and the secretion of IFN-gamma. The expression of MHC class II molecule in macrophages at the early stage of Y 17XL infection was significantly increased, accompanied by a significant increase in the number of macrophages, suggesting that macrophages were effectively activated and activated by IFN-gamma stimulation.
It has been proved that macrophages are an important source of non-specific immune effector molecule NO. Nitrogen peroxide formed after NO oxidation is the main effector molecule for macrophages to exert cytotoxicity and protozoan growth inhibition during the erythrocytic phase. Although the role of NO in the immune mechanism of anti-erythrocytic phase Plasmodium It is controversial, but our previous studies have found that in the early stage of Plasmodium yoelii infection, with the increase of nitric oxide synthesis in DBA/2 mice, the rising trend of erythrocyte infection rate was restrained, and many merozoite attachments on reticulocyte surface were observed. The results showed that although the macrophages of DBA/2 mice did not show phagocytosis at the first 4 days after infection, the NO level in the supernatant of spleen cells increased significantly at the third day after infection. Meanwhile, the level of protozoaemia increased slowly, suggesting that malaria infection was present. Early macrophages can inhibit protozoa by secreting NO.
Non-specific phagocytosis of macrophages includes two mechanisms: irrational phagocytosis and regulatory phagocytosis. Irregular phagocytosis of macrophages is accomplished by recognizing pathogen surface pattern recognition receptors including scavenger receptors and mannose receptors. CD36 is a scavenger receptor family B-type receptor. It is currently believed that CD36 mediates macrophage non-regulation. Some studies have shown that the binding of CD36 to erythrocytes infected by Plasmodium falciparum affects the severity of malaria. The deficiency of CD36 expression is closely related to severe malaria, especially cerebral malaria. The phagocytic activity of human and mouse macrophages was markedly increased, while that of anti-CD36 monoclonal antibody was decreased. Receptor blockade and CD36 gene knockout studies showed that both human and mouse macrophages could phagocytize erythrocytes (RPEs) infected by Plasmodium in the circular phase in vitro, depending on macrophages. CD36 molecules on the surface, rather than other surface receptors (ICAM-1, alpha v beta 3, or PECAM-1 or pattern recognition receptors such as mannose receptors or CD14), also play a key role in innate immune defense against P.falciparum gametophyte clearance by monocytes and macrophages. During the natural infection of mice, the expression of CD36 on the surface of macrophages began to increase significantly from the 3rd day after infection, and the phagocytic capacity of corresponding macrophages increased significantly with the peak of CD36 expression on the 10th day after infection.
The protective immune response in the erythrocytic phase of malaria passes through the acute phase of infection and is eventually cleared of the parasite by a specific antibody-mediated immune response. The antibody-mediated regulatory phagocytosis may be one of the important mechanisms of the acquired immune response in the erythrocytic phase of malaria infection. FcR-mediated regulatory phagocytosis is the key to eliminating P.berghei XAT intraerythrocytic infection, suggesting that phagocytic activity of splenic macrophages is essential for host resistance to malaria infection. FcR gamma-/-(gamma chain knockout) mice are susceptible to P.berghei XAT due to lack of antibody-dependent phagocytosis and antibody-dependent cytotoxicity (ADCC). Mota et al. confirmed that the peripheral blood macrophages of CBA mice could exert antibody-mediated regulatory phagocytosis against P. chabaudi-chabaudi-infected erythrocytes and possess the specificity of the strain. Our previous study showed that DBA/2 mice had specific IgG against P. chabaudi-infected erythrocytes on the 10th day after the deadly P. yoelii infection. The expression of Fc gamma R I (CD64) on the surface of macrophages began to increase from the 5th day after infection, then gradually increased and maintained a high level. At the same time, the phagocytosis rate of macrophages was at a high level from the 8th to 15th day after infection. Macrophages may play a regulatory phagocytosis role through CD64.
In conclusion, this study systematically and dynamically observed the activation and effect of macrophages in resistant DBA/2 mice during natural infection of P.y 17XL, and further revealed that macrophages play an important role in both innate and acquired immunity against malaria infection. Further elucidation of the relevant mechanisms will provide necessary theoretical and experimental basis for the development of effective malaria vaccines and new antimalarial drugs.
conclusion
Macrophages play an important role in both innate and acquired immunity in the process of lethal Plasmodium yoelii infection.
The number of splenic macrophages in 1.DBA / 2 mice increased gradually.
2. the expression of MHC class II molecules indicated that macrophages were activated effectively under the stimulation of IFN- gamma.
3. The expression of scavenger receptor CD36 in macrophages increased significantly in the early stage of infection, which mediated the irrational phagocytosis.
4. Fc gamma RI (CD64), as a macrophage expression, increased significantly in the late stage of infection, which may be involved in antibody-mediated regulatory phagocytosis.
5. The level of NO secreted by macrophages increased significantly in the early stage of infection, thus inhibiting the outbreak of Plasmodium.
【學位授予單位】：中國醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R392
本文編號：2212245
[Abstract]:Preface
Malaria is a global concern and a high incidence of infectious diseases. According to the latest report from Nature, the number of people infected with malaria in the world reached 515 million by 2002, and the number of people threatened worldwide reached 2.2 billion. It is an important strategy to effectively control the occurrence and development of malaria, and its necessary prerequisite depends on the further elucidation of the host protective immune response mechanism.
Monocyte-macrophage plays a significant role in the infection process of Plasmodium in different aspects. It can not only act as a non-specific immune system cell to perform non-specific phagocytosis and kill, but also participate in the mediation of specific immune response and play an important role in antigen presentation and immune regulation. It is characterized by the orderly activation of Th1 and Th2 immune responses, with the participation of CD_4~+Yh cells and antibodies. In the acute phase of infection, IFN-gamma-dominated Th1 inflammatory cytokines activate immune effector cells such as macrophages, inhibit the explosive proliferation of Plasmodium in the intraerythrocytic phase, effectively control the rapid rise of protozoemia: the subsequent response to Th Our previous studies have shown that activated monocytes and macrophages may be one of the key factors to control the level of parasitemia in the early stage of Plasmodium yoelii infection. Further explore and clarify.
In this study, P.y 17XL-resistant DBA/2 mice were selected as the research objects. The changes of membrane molecules, NO content and phagocytic function of macrophages were observed for the first time. The aim of this study was to further clarify the activation rules and effect characteristics of macrophages in malaria infection, and to develop effective malaria vaccines and new antimalarial drugs. Development provides new theoretical and experimental basis.
Experimental materials and methods
In this study, P.y 17XL-resistant DBA/2 mice were selected as the research objects.Giemsa thin blood membrane staining and light microscopy were used to count the infection rate of red blood cells.Flow cytometry was used to detect the number of splenic macrophages and the expression levels of surface membrane molecules CD36, CD64 and MHC II during P.y 17XL infection in DBA/2 mice. The content of NO_2~- secreted by splenic macrophages and the phagocytic function of macrophages were examined by microscope.
experimental result
In DBA/2 mice, the level of protozomia increased slowly after P.y 17XL attack, decreased gradually after 7 days of infection, and all the erythrocytes infected by P.falciparum were cleared after 15 days of infection. In the early stage of acute infection (3-5 days), the level of NO secreted by splenic macrophages increased significantly, and then the phagocytic capacity of macrophages increased. The phagocytosis rate was as high as 95% on the 5th day after infection, and then remained at a high level on the 10th day after infection; the MHC class II molecule and CD36 on the surface of splenic macrophages began to increase on the 3rd day after infection and reached their peak value on the 10th day after infection; the expression of CD64 began to increase on the 5th day after infection, and then remained at a high level. The number of macrophages also increased significantly at 3D after infection and reached the peak at 10d.
discuss
In recent years, many studies have shown that innate and acquired immune responses participate in the control of erythrocytic malaria infection, reduce the burden of parasites, and delay the development of severe malaria infection. Previous studies have shown that monocytes circulating in body fluid and macrophages in tissues (liver, spleen) can participate in the control of malaria infection by eliminating the erythrocytes (PRBC) infected by Plasmodium. Our previous studies have also confirmed that CD_4~+Th1 cells of resistant DBA/2 mice can produce high levels of IFN-gamma in the early stage of infection, and activated macrophages possess high levels of IFN-gamma. In this study, the activation of macrophages during P.y 17XL infection was systematically observed in a P.y 17XL resistant DBA/2 mouse model. The results showed that macrophages were activated rapidly in the early stage of P.y 17XL infection in order to produce secretory effects. The molecule NO inhibits protozoa and non-specific phagocytosis to kill Plasmodium.
Macrophages are important effector cells in DBA / 2 mice producing antimalarial protective immune responses.
Previous studies have shown that as full-time antigen presenting cells, macrophages can present antigens to T cells by expressing MHC molecules and costimulatory molecules, providing necessary activation signals for T cell activation and controlling the direction of T cell differentiation, thereby affecting the outcome of malaria infection. MHC class II molecules are important markers of macrophage activation. The protective immunity of non-lethal Plasmodium yoelii and Plasmodium charantii during intraerythrocytic infection was completely dependent on MHC class II molecules. The expression of MHC class II and CD80 in macrophages of BALB/c mice infected with non-lethal Plasmodium yoelii increased significantly, thus promoting the activation of T cells and the secretion of IFN-gamma. The expression of MHC class II molecule in macrophages at the early stage of Y 17XL infection was significantly increased, accompanied by a significant increase in the number of macrophages, suggesting that macrophages were effectively activated and activated by IFN-gamma stimulation.
It has been proved that macrophages are an important source of non-specific immune effector molecule NO. Nitrogen peroxide formed after NO oxidation is the main effector molecule for macrophages to exert cytotoxicity and protozoan growth inhibition during the erythrocytic phase. Although the role of NO in the immune mechanism of anti-erythrocytic phase Plasmodium It is controversial, but our previous studies have found that in the early stage of Plasmodium yoelii infection, with the increase of nitric oxide synthesis in DBA/2 mice, the rising trend of erythrocyte infection rate was restrained, and many merozoite attachments on reticulocyte surface were observed. The results showed that although the macrophages of DBA/2 mice did not show phagocytosis at the first 4 days after infection, the NO level in the supernatant of spleen cells increased significantly at the third day after infection. Meanwhile, the level of protozoaemia increased slowly, suggesting that malaria infection was present. Early macrophages can inhibit protozoa by secreting NO.
Non-specific phagocytosis of macrophages includes two mechanisms: irrational phagocytosis and regulatory phagocytosis. Irregular phagocytosis of macrophages is accomplished by recognizing pathogen surface pattern recognition receptors including scavenger receptors and mannose receptors. CD36 is a scavenger receptor family B-type receptor. It is currently believed that CD36 mediates macrophage non-regulation. Some studies have shown that the binding of CD36 to erythrocytes infected by Plasmodium falciparum affects the severity of malaria. The deficiency of CD36 expression is closely related to severe malaria, especially cerebral malaria. The phagocytic activity of human and mouse macrophages was markedly increased, while that of anti-CD36 monoclonal antibody was decreased. Receptor blockade and CD36 gene knockout studies showed that both human and mouse macrophages could phagocytize erythrocytes (RPEs) infected by Plasmodium in the circular phase in vitro, depending on macrophages. CD36 molecules on the surface, rather than other surface receptors (ICAM-1, alpha v beta 3, or PECAM-1 or pattern recognition receptors such as mannose receptors or CD14), also play a key role in innate immune defense against P.falciparum gametophyte clearance by monocytes and macrophages. During the natural infection of mice, the expression of CD36 on the surface of macrophages began to increase significantly from the 3rd day after infection, and the phagocytic capacity of corresponding macrophages increased significantly with the peak of CD36 expression on the 10th day after infection.
The protective immune response in the erythrocytic phase of malaria passes through the acute phase of infection and is eventually cleared of the parasite by a specific antibody-mediated immune response. The antibody-mediated regulatory phagocytosis may be one of the important mechanisms of the acquired immune response in the erythrocytic phase of malaria infection. FcR-mediated regulatory phagocytosis is the key to eliminating P.berghei XAT intraerythrocytic infection, suggesting that phagocytic activity of splenic macrophages is essential for host resistance to malaria infection. FcR gamma-/-(gamma chain knockout) mice are susceptible to P.berghei XAT due to lack of antibody-dependent phagocytosis and antibody-dependent cytotoxicity (ADCC). Mota et al. confirmed that the peripheral blood macrophages of CBA mice could exert antibody-mediated regulatory phagocytosis against P. chabaudi-chabaudi-infected erythrocytes and possess the specificity of the strain. Our previous study showed that DBA/2 mice had specific IgG against P. chabaudi-infected erythrocytes on the 10th day after the deadly P. yoelii infection. The expression of Fc gamma R I (CD64) on the surface of macrophages began to increase from the 5th day after infection, then gradually increased and maintained a high level. At the same time, the phagocytosis rate of macrophages was at a high level from the 8th to 15th day after infection. Macrophages may play a regulatory phagocytosis role through CD64.
In conclusion, this study systematically and dynamically observed the activation and effect of macrophages in resistant DBA/2 mice during natural infection of P.y 17XL, and further revealed that macrophages play an important role in both innate and acquired immunity against malaria infection. Further elucidation of the relevant mechanisms will provide necessary theoretical and experimental basis for the development of effective malaria vaccines and new antimalarial drugs.
conclusion
Macrophages play an important role in both innate and acquired immunity in the process of lethal Plasmodium yoelii infection.
The number of splenic macrophages in 1.DBA / 2 mice increased gradually.
2. the expression of MHC class II molecules indicated that macrophages were activated effectively under the stimulation of IFN- gamma.
3. The expression of scavenger receptor CD36 in macrophages increased significantly in the early stage of infection, which mediated the irrational phagocytosis.
4. Fc gamma RI (CD64), as a macrophage expression, increased significantly in the late stage of infection, which may be involved in antibody-mediated regulatory phagocytosis.
5. The level of NO secreted by macrophages increased significantly in the early stage of infection, thus inhibiting the outbreak of Plasmodium.
【學位授予單位】：中國醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R392
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