小神经胶质细胞通过产生胞外陷阱捕获并杀伤单增性李斯特菌分子机制初步研究

发布时间：2018-06-08 01:46

本文选题：小神经胶质细胞 + 胞外陷阱　；参考：《吉林大学》2015年硕士论文

【摘要】：胞外陷阱（extracellular traps，ETs）最早发现于嗜中性粒细胞中，是免疫系统一种独特的防御病原菌的方式。ETs是先天性免疫细胞受到病原菌或一些化学物质如佛波醇（phorbol-12-myristate-13-acetate，PMA）的刺激释放的，一种浓缩染色质折叠成的纤维状结构，另有颗粒衍生肽和一些胞内蛋白装饰其上。除了嗜中性粒细胞，肥大细胞、嗜酸性粒细胞和巨噬细胞都可以产生ETs，而巨噬细胞产生的胞外陷阱称为METs。NETs的形成是一种复杂的过程，NADPH氧化酶的活化和Raf-MEK-ERK通路的感应是其中最重要的途径。人们针对NETs已经做了大量的研究，但对于与它同源的先天免疫细胞——巨噬细胞产生胞外陷阱的机制了解依然相对较少。小神经胶质细胞是中枢神经系统（CNS）的常驻巨噬细胞，它们大量存在于中枢神经系统中，占中枢神经系统总细胞量的百分之十二左右。小神经胶质细胞在大脑中起着监测神经系统的作用，具有对抗病原体入侵的能力。而产单核细胞增生性李斯特氏菌（Listeria monocytogenes，Lm）就是一种可穿过血脑屏障，在脑中生长繁殖的菌种。在此之前，我们发现了人巨噬细胞系THP-1及小鼠巨噬细胞系RAW264.7在Lm的侵染下可以产生METs，而具有侵袭神经系统功能的Lm是否能引起小神经胶质细胞（神经系统的巨噬细胞）产生METs，，目前国内外还没有报道。小神经胶质细胞株BV-2具有小神经胶质细胞的形态和功能特征，且已经被证明是小神经胶质细胞有效的、主要的替代株。本文以BV-2为模式细胞，对Lm及PMA能否诱导BV-2产生胞外陷阱及其相关机制进行初步研究。首先，我们利用荧光显微镜观察BV-2细胞在PMA及Lm（ATCC19111）诱导下产生METs的组成成分；利用荧光酶标仪检测不同时间和菌量作用下BV-2产生的胞外DNA的量，并利用sytox green染色方法进一步验证时间和菌量对胞外陷阱产量的影响；利用活/死菌染色、菌落计数的方法检测BV-2产生的METs对Lm的杀伤作用。结果显示PMA及Lm可以诱导BV-2产生胞外陷阱，且该METs中含有髓过氧化物酶、组蛋白H3和弹力蛋白酶成分；此种METs的产生与时间和菌量呈正相关；Lm感染BV-2产生的METs有一定的杀菌效果。其次，利用免疫印迹技术和胞外DNA定量方法验证BV-2胞外陷阱的形成与Raf-MEK-ERK通路、NADPH氧化酶的关联性；利用HE染色、免疫荧光技术和DNA胞外定量技术验证METs模型在大鼠脑内是否存在。结果显示ERK通路、NADPH氧化酶和ROS的活化均参与Lm诱导BV-2形成METs的过程；感染Lm的大鼠脑内有METs的存在，且脑脊液中DNA的含量也明显增多。本研究初步探讨了Lm及PMA激活小神经胶质细胞产生胞外陷阱的分子机制，为巨噬细胞及小神经胶质细胞胞外陷阱的研究提供了理论依据，同时也为Lm导致的神经性疾病的科学研究和临床治疗奠定基础。
[Abstract]:Extracellular traps ETs were first found in neutrophils. ETs are a unique way of defense against pathogens in the immune system. ETs are released by innate immune cells stimulated by pathogenic bacteria or some chemicals such as phorbol-12-Eritrestate-13-acetatePMA. A fibrous structure folded into condensed chromatin, adorned with granular derived peptides and some intracellular proteins. Except for neutrophilic granulocytes, mast cells, Eosinophilic granulocytes and macrophages can produce ETs. The extracellular trap produced by macrophages called METs.NETs is a complex process of activation of NADPH oxidase and induction of Raf-MEK-ERK pathway. A great deal of research has been done on NETs, but there is still relatively little understanding of the mechanism of extracellular traps produced by innate immune cells-macrophages that are homologous to NETs. Small glial cells are resident macrophages of the central nervous system (CNS). They are abundant in the central nervous system, accounting for about 12% of the total number of central nervous system cells. Small glial cells play a role in the monitoring of the nervous system in the brain and have the ability to counteract the invasion of pathogens. Listeria monocytogenes (Lm) is a species that crosses the blood-brain barrier to grow and reproduce in the brain. We found that human macrophage cell line THP-1 and mouse macrophage cell line RAW264.7 can produce METsunder the infection of LM, and whether Lm, which has the function of invading the nervous system, can induce the production of microglial cells (macrophages of the nervous system) Mets, at present there are no reports at home and abroad. The microglial cell line BV-2 has the morphological and functional characteristics of microglia and has been proved to be an effective and main substitute for microglia. In this paper, we studied whether Lm and PMA could induce BV-2 to produce extracellular traps and their related mechanisms. Firstly, we observed the components of Mets induced by PMA and LmACC19111 by fluorescence microscope. The amount of extracellular DNA produced by BV-2 at different time and amount of bacteria was detected by fluorescence enzyme marker, and the effect of time and quantity on the yield of extracellular trap was further verified by sytox green staining. The killing effect of Mets produced by BV-2 on LM was detected by colony counting method. The results showed that PMA and LM could induce BV-2 to produce extracellular traps, and the Mets contained myeloperoxidase, histone H3 and elastase. The production of Mets was positively correlated with the time and quantity of BV-2. Secondly, the relationship between the formation of extracellular traps of BV-2 and Raf-MEK-ERK pathway and NADPH oxidase was verified by Western blotting and quantitative analysis of extracellular DNA. The presence of Mets in rat brain was verified by HE staining, immunofluorescence and DNA quantitative analysis. The results showed that the activation of NADPH oxidase and Ros in ERK pathway was involved in the formation of Mets in Lm-induced BV-2, and the presence of Mets in the brain of rats infected with Lm. The molecular mechanism of Lm and PMA activating microglial cells to produce extracellular traps is discussed, which provides a theoretical basis for the study of extracellular traps in macrophages and microglial cells. It also lays the foundation for the scientific research and clinical treatment of the neurological diseases caused by LM.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

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