約氏瘧原蟲來源的巨噬細(xì)胞遷移抑制因子同源分子的功能研究
發(fā)布時(shí)間：2018-09-10 15:02
【摘要】：宿主來源的MIF(Macrophage Migration Inhibitory Factor)分子被發(fā)現(xiàn)在瘧疾感染病理、特別是貧血中發(fā)揮了重要作用。近年來,包括本實(shí)驗(yàn)室在內(nèi)的三個(gè)研究小組先后報(bào)道了兩個(gè)瘧原蟲來源的MIF分子,Plasmodium falciparum MIF(PfMIF)和P.berghei MIF(PbMIF)的鑒定和初步功能探討。研究發(fā)現(xiàn),瘧原蟲來源的MIF分子具有調(diào)節(jié)宿主免疫細(xì)胞的活性。然而,瘧原蟲MIF分子的調(diào)節(jié)機(jī)制、特別是它和宿主MIF的聯(lián)合效應(yīng)仍然不清楚。本實(shí)驗(yàn)室前期工作還鑒定了另一個(gè)瘧原蟲種來源的MIF分子——P.yoelii MIF(PyMIF),并對其結(jié)構(gòu)和初步活性進(jìn)行了研究,盡管PyMIF與小鼠MIF分子的氨基酸序列同源性僅為30%左右,但是二者的晶體解析結(jié)構(gòu)高度相似。本論文在實(shí)驗(yàn)室前期工作的基礎(chǔ)上,著重探討PyMIF對靶細(xì)胞的調(diào)節(jié)機(jī)制,包括其細(xì)胞膜受體、是否能夠內(nèi)吞進(jìn)入細(xì)胞、調(diào)節(jié)的信號通路、下游分子、全細(xì)胞效應(yīng)、凋亡機(jī)制以及動(dòng)物模型研究等幾個(gè)方面。 本論文的研究工作發(fā)現(xiàn)小鼠纖維細(xì)胞和巨噬細(xì)胞都是PyMIF的靶細(xì)胞,并且PyMIF在調(diào)節(jié)信號通路方面與宿主小鼠MIF(MmMIF)相似,它可以活化MAPK/ERK、PI3K/AKT信號通路,并抑制AP-1活化,但對JAK/STAT和NF-κB信號通路沒有調(diào)節(jié)作用;重要的是,盡管二者活化信號通路的作用相似,但他們的聯(lián)合作用卻呈現(xiàn)低濃度活化、高濃度抑制的復(fù)雜效應(yīng)。同時(shí),盡管PyMIF和MmMIF都抑制巨噬細(xì)胞凋亡,但在機(jī)制上也不完全相同;不僅如此,PyMIF明顯調(diào)節(jié)了更多下游信號分子的轉(zhuǎn)錄。在膜受體和內(nèi)吞研究方面,由于受體表達(dá)或純化的困難以及蛋白標(biāo)記方法沒有成功,這部分研究工作目前尚未完成。 在小鼠模型研究中,在不同品系小鼠感染瘧原蟲后均可在外周血檢測到蟲源MIF,并證明蟲源MIF水平與感染蟲血率相關(guān);并且,尾靜脈注射PyMIF導(dǎo)致感染過程的延長和蟲血率波動(dòng),并且上調(diào)了TNF-α和IL-6細(xì)胞因子水平;但對感染小鼠配子體出現(xiàn)時(shí)間和數(shù)目并無影響。另外,在感染后第6天給藥治療小鼠后,小鼠外周血PyMIF水平先上升一天后迅速下降,至第三天完全消失,為流行地區(qū)現(xiàn)場樣本采集的時(shí)間點(diǎn)提供了依據(jù),也間接提示蟲源MIF有可能作為病程指標(biāo)。 最后,本研究還比較了三種MIF內(nèi)毒素去除方法,證明本實(shí)驗(yàn)室優(yōu)化的C8反相柱—非復(fù)性法是一種簡單易行、可實(shí)驗(yàn)室規(guī)模應(yīng)用的有效去除少量蛋白質(zhì)內(nèi)毒素的方法,為功能研究提供了重要的技術(shù)支持。 本論文工作對瘧原蟲MIF作用機(jī)制研究方面提供了重要線索,而體內(nèi)研究數(shù)據(jù)又為同期進(jìn)行的現(xiàn)場樣本采集和數(shù)據(jù)分析工作提供了重要的借鑒,因此本論文工作不僅有助于深入理解瘧原蟲逃逸宿主免疫反應(yīng)和感染的機(jī)理,并對這些方面的研究提供了新的思路,也為本實(shí)驗(yàn)室進(jìn)一步研究工作奠定了重要基礎(chǔ)。
[Abstract]:Host-derived MIF (Macrophage Migration Inhibitory Factor) molecules have been found to play an important role in the pathogenesis of malaria infection, especially anemia. In recent years, three research groups, including our laboratory, have reported the identification and preliminary functional study of two MIF molecules, Plasmodium falciparum MIF (PfMIF) and P.berghei MIF (PbMIF), from which Plasmodium falciparum MIF (PfMIF) and P.berghei MIF (PbMIF) were derived from two Plasmodium parasites. MIF molecules derived from Plasmodium falciparum have the activity of regulating host immune cells. However, the regulatory mechanism of Plasmodium MIF molecules, especially its combined effect with host MIF, remains unclear. In our laboratory, we also identified P.yoelii MIF (PyMIF), another plasmodium species, and studied its structure and preliminary activity, although the amino acid sequence homology between PyMIF and mouse MIF was only about 30%. However, their crystal structures are highly similar. Based on the previous work in laboratory, this paper focuses on the regulation mechanism of PyMIF on target cells, including its cell membrane receptor, whether it can endocytosis into cells, regulated signal pathways, downstream molecules, whole cell effects. The mechanism of apoptosis and the study of animal models. In this paper, we found that mouse fibroblasts and macrophages are both target cells of PyMIF, and PyMIF is similar to host mouse MIF (MmMIF) in regulating signal pathway, which can activate MAPK/ERK,PI3K/AKT signaling pathway and inhibit AP-1 activation. However, the JAK/STAT and NF- 魏 B signaling pathways were not regulated. It was important that, although the two activated signaling pathways had similar effects, their combined effects showed a complex effect of low concentration activation and high concentration inhibition. At the same time, both PyMIF and MmMIF inhibited macrophage apoptosis, but the mechanism was not the same. Not only that, PyMIF significantly regulated the transcription of more downstream signaling molecules. In the study of membrane receptor and endocytosis, the research work has not been completed because of the difficulty of expression or purification of receptor and the failure of protein labeling method. In the mouse model study, parasitogenic MIF, was detected in peripheral blood of different strains of mice infected with Plasmodium falciparum, and the level of insect-derived MIF was correlated with the blood rate of infected parasites, and tail vein injection of PyMIF resulted in prolonged infection process and fluctuating rate of parasite blood. The levels of TNF- 偽 and IL-6 cytokines were up-regulated, but there was no effect on the time and number of gametophytes in infected mice. In addition, after the mice were treated with drugs on the 6th day after infection, the level of PyMIF in peripheral blood of the mice first increased for one day, then decreased rapidly, and disappeared completely on the third day, which provided the basis for collecting samples in the epidemic area. It is also suggested that MIF may be used as an index of disease course. Finally, three kinds of MIF endotoxin removal methods were compared, which proved that the C8 reverse-column non-renaturation method optimized by our laboratory was a simple and effective method for removing a small amount of protein endotoxin on a laboratory scale. It provides important technical support for functional research. This work provides important clues for the study of the mechanism of Plasmodium MIF, and the in vivo research data provide important reference for the field sample collection and data analysis in the same period. Therefore, this work is not only helpful to understand the immune response and infection mechanism of Plasmodium parasites, but also provides new ideas for the study of these aspects, and lays an important foundation for further research in our laboratory.
【學(xué)位授予單位】：中國協(xié)和醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R362
本文編號：2234795
[Abstract]:Host-derived MIF (Macrophage Migration Inhibitory Factor) molecules have been found to play an important role in the pathogenesis of malaria infection, especially anemia. In recent years, three research groups, including our laboratory, have reported the identification and preliminary functional study of two MIF molecules, Plasmodium falciparum MIF (PfMIF) and P.berghei MIF (PbMIF), from which Plasmodium falciparum MIF (PfMIF) and P.berghei MIF (PbMIF) were derived from two Plasmodium parasites. MIF molecules derived from Plasmodium falciparum have the activity of regulating host immune cells. However, the regulatory mechanism of Plasmodium MIF molecules, especially its combined effect with host MIF, remains unclear. In our laboratory, we also identified P.yoelii MIF (PyMIF), another plasmodium species, and studied its structure and preliminary activity, although the amino acid sequence homology between PyMIF and mouse MIF was only about 30%. However, their crystal structures are highly similar. Based on the previous work in laboratory, this paper focuses on the regulation mechanism of PyMIF on target cells, including its cell membrane receptor, whether it can endocytosis into cells, regulated signal pathways, downstream molecules, whole cell effects. The mechanism of apoptosis and the study of animal models. In this paper, we found that mouse fibroblasts and macrophages are both target cells of PyMIF, and PyMIF is similar to host mouse MIF (MmMIF) in regulating signal pathway, which can activate MAPK/ERK,PI3K/AKT signaling pathway and inhibit AP-1 activation. However, the JAK/STAT and NF- 魏 B signaling pathways were not regulated. It was important that, although the two activated signaling pathways had similar effects, their combined effects showed a complex effect of low concentration activation and high concentration inhibition. At the same time, both PyMIF and MmMIF inhibited macrophage apoptosis, but the mechanism was not the same. Not only that, PyMIF significantly regulated the transcription of more downstream signaling molecules. In the study of membrane receptor and endocytosis, the research work has not been completed because of the difficulty of expression or purification of receptor and the failure of protein labeling method. In the mouse model study, parasitogenic MIF, was detected in peripheral blood of different strains of mice infected with Plasmodium falciparum, and the level of insect-derived MIF was correlated with the blood rate of infected parasites, and tail vein injection of PyMIF resulted in prolonged infection process and fluctuating rate of parasite blood. The levels of TNF- 偽 and IL-6 cytokines were up-regulated, but there was no effect on the time and number of gametophytes in infected mice. In addition, after the mice were treated with drugs on the 6th day after infection, the level of PyMIF in peripheral blood of the mice first increased for one day, then decreased rapidly, and disappeared completely on the third day, which provided the basis for collecting samples in the epidemic area. It is also suggested that MIF may be used as an index of disease course. Finally, three kinds of MIF endotoxin removal methods were compared, which proved that the C8 reverse-column non-renaturation method optimized by our laboratory was a simple and effective method for removing a small amount of protein endotoxin on a laboratory scale. It provides important technical support for functional research. This work provides important clues for the study of the mechanism of Plasmodium MIF, and the in vivo research data provide important reference for the field sample collection and data analysis in the same period. Therefore, this work is not only helpful to understand the immune response and infection mechanism of Plasmodium parasites, but also provides new ideas for the study of these aspects, and lays an important foundation for further research in our laboratory.
【學(xué)位授予單位】：中國協(xié)和醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R362
【引證文獻(xiàn)】
相關(guān)碩士學(xué)位論文 前1條
1 師文娟;人Ⅱ型CD74及人源與瘧原蟲來源MIF在昆蟲細(xì)胞中的表達(dá)研究[D];中國協(xié)和醫(yī)科大學(xué);2010年
，本文編號：2234795
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