本文選題：小神經(jīng)膠質(zhì)細(xì)胞 + 胞外陷阱　； 參考：《吉林大學(xué)》2015年碩士論文

【摘要】：胞外陷阱（extracellular traps，ETs）最早發(fā)現(xiàn)于嗜中性粒細(xì)胞中，是免疫系統(tǒng)一種獨(dú)特的防御病原菌的方式。ETs是先天性免疫細(xì)胞受到病原菌或一些化學(xué)物質(zhì)如佛波醇（phorbol-12-myristate-13-acetate，PMA）的刺激釋放的，一種濃縮染色質(zhì)折疊成的纖維狀結(jié)構(gòu)，另有顆粒衍生肽和一些胞內(nèi)蛋白裝飾其上。除了嗜中性粒細(xì)胞，肥大細(xì)胞、嗜酸性粒細(xì)胞和巨噬細(xì)胞都可以產(chǎn)生ETs，而巨噬細(xì)胞產(chǎn)生的胞外陷阱稱為METs。NETs的形成是一種復(fù)雜的過程，NADPH氧化酶的活化和Raf-MEK-ERK通路的感應(yīng)是其中最重要的途徑。人們針對(duì)NETs已經(jīng)做了大量的研究，但對(duì)于與它同源的先天免疫細(xì)胞——巨噬細(xì)胞產(chǎn)生胞外陷阱的機(jī)制了解依然相對(duì)較少。 小神經(jīng)膠質(zhì)細(xì)胞是中樞神經(jīng)系統(tǒng)（CNS）的常駐巨噬細(xì)胞，它們大量存在于中樞神經(jīng)系統(tǒng)中，占中樞神經(jīng)系統(tǒng)總細(xì)胞量的百分之十二左右。小神經(jīng)膠質(zhì)細(xì)胞在大腦中起著監(jiān)測(cè)神經(jīng)系統(tǒng)的作用，具有對(duì)抗病原體入侵的能力。而產(chǎn)單核細(xì)胞增生性李斯特氏菌（Listeria monocytogenes，Lm）就是一種可穿過血腦屏障，在腦中生長繁殖的菌種。 在此之前，我們發(fā)現(xiàn)了人巨噬細(xì)胞系THP-1及小鼠巨噬細(xì)胞系RAW264.7在Lm的侵染下可以產(chǎn)生METs，而具有侵襲神經(jīng)系統(tǒng)功能的Lm是否能引起小神經(jīng)膠質(zhì)細(xì)胞（神經(jīng)系統(tǒng)的巨噬細(xì)胞）產(chǎn)生METs，，目前國內(nèi)外還沒有報(bào)道。小神經(jīng)膠質(zhì)細(xì)胞株BV-2具有小神經(jīng)膠質(zhì)細(xì)胞的形態(tài)和功能特征，且已經(jīng)被證明是小神經(jīng)膠質(zhì)細(xì)胞有效的、主要的替代株。本文以BV-2為模式細(xì)胞，對(duì)Lm及PMA能否誘導(dǎo)BV-2產(chǎn)生胞外陷阱及其相關(guān)機(jī)制進(jìn)行初步研究。 首先，我們利用熒光顯微鏡觀察BV-2細(xì)胞在PMA及Lm（ATCC19111）誘導(dǎo)下產(chǎn)生METs的組成成分；利用熒光酶標(biāo)儀檢測(cè)不同時(shí)間和菌量作用下BV-2產(chǎn)生的胞外DNA的量，并利用sytox green染色方法進(jìn)一步驗(yàn)證時(shí)間和菌量對(duì)胞外陷阱產(chǎn)量的影響；利用活/死菌染色、菌落計(jì)數(shù)的方法檢測(cè)BV-2產(chǎn)生的METs對(duì)Lm的殺傷作用。結(jié)果顯示PMA及Lm可以誘導(dǎo)BV-2產(chǎn)生胞外陷阱，且該METs中含有髓過氧化物酶、組蛋白H3和彈力蛋白酶成分；此種METs的產(chǎn)生與時(shí)間和菌量呈正相關(guān)；Lm感染BV-2產(chǎn)生的METs有一定的殺菌效果。 其次，利用免疫印跡技術(shù)和胞外DNA定量方法驗(yàn)證BV-2胞外陷阱的形成與Raf-MEK-ERK通路、NADPH氧化酶的關(guān)聯(lián)性；利用HE染色、免疫熒光技術(shù)和DNA胞外定量技術(shù)驗(yàn)證METs模型在大鼠腦內(nèi)是否存在。結(jié)果顯示ERK通路、NADPH氧化酶和ROS的活化均參與Lm誘導(dǎo)BV-2形成METs的過程；感染Lm的大鼠腦內(nèi)有METs的存在，且腦脊液中DNA的含量也明顯增多。 本研究初步探討了Lm及PMA激活小神經(jīng)膠質(zhì)細(xì)胞產(chǎn)生胞外陷阱的分子機(jī)制，為巨噬細(xì)胞及小神經(jīng)膠質(zhì)細(xì)胞胞外陷阱的研究提供了理論依據(jù)，同時(shí)也為Lm導(dǎo)致的神經(jīng)性疾病的科學(xué)研究和臨床治療奠定基礎(chǔ)。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.61

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