约氏疟原虫来源的巨噬细胞迁移抑制因子同源分子的功能研究
发布时间：2018-09-10 15:02
【摘要】：宿主来源的MIF(Macrophage Migration Inhibitory Factor)分子被发现在疟疾感染病理、特别是贫血中发挥了重要作用。近年来,包括本实验室在内的三个研究小组先后报道了两个疟原虫来源的MIF分子,Plasmodium falciparum MIF(PfMIF)和P.berghei MIF(PbMIF)的鉴定和初步功能探讨。研究发现,疟原虫来源的MIF分子具有调节宿主免疫细胞的活性。然而,疟原虫MIF分子的调节机制、特别是它和宿主MIF的联合效应仍然不清楚。本实验室前期工作还鉴定了另一个疟原虫种来源的MIF分子——P.yoelii MIF(PyMIF),并对其结构和初步活性进行了研究,尽管PyMIF与小鼠MIF分子的氨基酸序列同源性仅为30%左右,但是二者的晶体解析结构高度相似。本论文在实验室前期工作的基础上,着重探讨PyMIF对靶细胞的调节机制,包括其细胞膜受体、是否能够内吞进入细胞、调节的信号通路、下游分子、全细胞效应、凋亡机制以及动物模型研究等几个方面。 本论文的研究工作发现小鼠纤维细胞和巨噬细胞都是PyMIF的靶细胞,并且PyMIF在调节信号通路方面与宿主小鼠MIF(MmMIF)相似,它可以活化MAPK/ERK、PI3K/AKT信号通路,并抑制AP-1活化,但对JAK/STAT和NF-κB信号通路没有调节作用;重要的是,尽管二者活化信号通路的作用相似,但他们的联合作用却呈现低浓度活化、高浓度抑制的复杂效应。同时,尽管PyMIF和MmMIF都抑制巨噬细胞凋亡,但在机制上也不完全相同;不仅如此,PyMIF明显调节了更多下游信号分子的转录。在膜受体和内吞研究方面,由于受体表达或纯化的困难以及蛋白标记方法没有成功,这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疟原虫后均可在外周血检测到虫源MIF,并证明虫源MIF水平与感染虫血率相关;并且,尾静脉注射PyMIF导致感染过程的延长和虫血率波动,并且上调了TNF-α和IL-6细胞因子水平;但对感染小鼠配子体出现时间和数目并无影响。另外,在感染后第6天给药治疗小鼠后,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,为流行地区现场样本采集的时间点提供了依据,也间接提示虫源MIF有可能作为病程指标。 最后,本研究还比较了三种MIF内毒素去除方法,证明本实验室优化的C8反相柱—非复性法是一种简单易行、可实验室规模应用的有效去除少量蛋白质内毒素的方法,为功能研究提供了重要的技术支持。 本论文工作对疟原虫MIF作用机制研究方面提供了重要线索,而体内研究数据又为同期进行的现场样本采集和数据分析工作提供了重要的借鉴,因此本论文工作不仅有助于深入理解疟原虫逃逸宿主免疫反应和感染的机理,并对这些方面的研究提供了新的思路,也为本实验室进一步研究工作奠定了重要基础。
[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- 伪 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.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R362
本文编号：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- 伪 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.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R362
【引证文献】
相关硕士学位论文 前1条
1 师文娟;人Ⅱ型CD74及人源与疟原虫来源MIF在昆虫细胞中的表达研究[D];中国协和医科大学;2010年
，本文编号：2234795
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