烟曲霉感染中TLR2和转录因子PU.1对Dectin-1表达影响的研究

发布时间：2018-05-27 16:01

本文选题：烟曲霉 + HBE细胞　；参考：《第二军医大学》2013年硕士论文

【摘要】：近年来随着易感人群的增加，烟曲霉感染的发病率呈现不断上升趋势。即使目前抗真菌药物研究取得很大进步，且临床诊疗水平不断提高，但侵袭性曲霉病（invasiveaspergillosis，IA）的病死率仍高达50%～90%。烟曲霉感染的研究证明，机体免疫状态是宿主发病的重要因素。随着研究的不断深入，模式识别受体（pattern recognitionreceptors，PRRs）在机体免疫调节中的作用日渐受到重视。人们认识到PRRs对病原体相关分子模式（pathogen-associated molecular patterns，PAMPs）的识别是宿主抗真菌免疫的始动环节。深入研究PRRs的作用及其调节机制，为从干预宿主免疫状态角度找到曲霉病防治新思路提供了可能。在抗真菌免疫中，Dectin-1是起主要作用的PRRs之一。Dectin-1是一种糖基化Ⅱ型跨膜受体，属于C型植物凝集素家族，主要通过识别β-(1,3)葡聚糖介导宿主抗真菌免疫防御反应。在烟曲霉感染中Dectin-1通过识别烟曲霉胞壁的β-(1,3)葡聚糖成份促进机体的保护性应答反应，包括对烟曲霉的摄取及杀伤（通过呼吸爆发介导），产生大量的细胞因子和趋化因子，包括肿瘤坏死因子（TNF）、白细胞介素1（IL-1）、白细胞介素6（IL-6）以及粒细胞-单核细胞集落刺激因子（GM-CSF）等。深入了解烟曲霉感染中Dectin-1的调节及作用机制，对研究烟曲霉发病及临床防治具有重要意义。本研究通过建立烟曲霉感染的体外模型，观察Dectin-1在烟曲霉感染后的表达变化，并通过siRNA沉默技术在不同水平评价Toll样受体2（TLR2）、转录因子PU.1对Dectin-1表达调控的影响。第一部分烟曲霉感染时TLR2对Dectin-1表达的影响目的：建立烟曲霉感染人支气管上皮细胞（human bronchial epithelial cells，HBE细胞）的体外模型，观察Dectin-1、TLR2表达变化以及二者之间的关系，为进一步研究不同PRRs之间的协同机制提供依据。方法：①烟曲霉孢子感染HBE细胞，感染复数（multiplicity of infection，MOI）为1。分别在0、6、18、24h终止感染，提取细胞总RNA、总蛋白，通过Western Blot、PCR方法分别在蛋白、mRNA水平上检测Dectin-1和TLR2的表达情况；②沉默TLR2，复制感染模型，通过Western Blot及流式细胞技术观察Dectin-1的表达变化。结果：①HBE细胞感染烟曲霉孢子6h后，Dectin-1的mRNA表达水平显著升高，差异具有统计学意义（P0.05），在感染后24h仍维持在较高水平；在HBE细胞静息状态下Dectin-1蛋白基础表达水平较低，与初始状态相比，，感染后6h表达量增加11倍（P0.01）。②TLR2的mRNA和蛋白在静息状态下的HBE细胞中即有较高水平表达，随着感染时间的延长，其蛋白表达水平有增高的趋势，但与静息状态下表达水平相比差异无统计学意义（P0.05）。③沉默TLR2，在烟曲霉感染18h时，HBE细胞中Dectin-1的表达受到显著抑制，差异具有统计学意义（P0.05）。结论：在烟曲霉感染中，HBE细胞内Dectin-1表达增强，而TLR2表达无明显变化；TLR2对Dectin-1的表达有协同作用，可能参与其上调表达过程，但具体的调节机制还不清楚。第二部分烟曲霉感染时转录因子PU.1对Dectin-1的调节作用目的：构建THP-1（人急性白血病单核细胞株）巨噬细胞烟曲霉感染模型，探讨烟曲霉感染时转录因子PU.1对THP-1巨噬细胞吞噬作用的影响和Dectin-1转录水平的调节作用。方法：①烟曲霉孢子感染THP-1巨噬细胞，MOI=1。分别在0、12、24h终止感染，提取细胞总蛋白，通过Western Blot方法检测PU.1蛋白表达情况；②沉默PU.1，复制THP-1巨噬细胞感染模型，Western Blot评价Dectin-1表达变化情况，激光共聚焦显微镜观察其对烟曲霉孢子吞噬能力的变化。结果：THP-1巨噬细胞在静息状态下能够表达转录因子PU.1，随着感染时间的延长其表达量逐渐增加，差异具有统计学意义（P0.05）；转录因子PU.1沉默后，在烟曲霉感染12h时，THP-1巨噬细胞中Dectin-1的表达显著抑制，差异具有统计学意义（P0.05）；转录因子PU.1沉默后，THP-1巨噬细胞对静息期、肿胀期烟曲霉孢子的吞噬能力下降。结论：在烟曲霉感染时转录因子PU.1表达上调，这影响了THP-1巨噬细胞的吞噬功能并可能在转录水平参与调控Dectin-1的表达，但是二者之间是否存在直接联系还需要进一步研究证实。
[Abstract]:In recent years, with the increase of susceptible population, the incidence of Aspergillus fumigatus infection is on the rise. Even at present, the study of anti fungal drugs has made great progress and the level of clinical diagnosis and treatment is increasing, but the mortality of invasive aspergillosis (invasiveaspergillosis, IA) is still up to 50% to 90%. Aspergillus fumigatus infection. State is an important factor in host disease. With the development of research, the role of pattern recognitionreceptors (PRRs) in immune regulation is becoming more and more important. It is recognized that the identification of PRRs to the pathogen associated molecular model (pathogen-associated molecular patterns, PAMPs) is the host antifungal immunity The role of PRRs and its regulatory mechanism are discussed. It is possible to find new ideas for prevention and treatment of aspergillosis from the point of view of intervening the host immune state.
In antifungal immunity, Dectin-1 is one of the main functions of PRRs.Dectin-1, a glycosylated type II transmembrane receptor, belonging to the C type plant lectin family, mainly mediated by the identification of beta (1,3) glucan to mediate the host antifungal immune defense response. In Aspergillus fumigatus infection, Dectin-1 is promoted by the identification of beta (1,3) glucan components of Aspergillus fumigatus wall. The protective response of the progressive organism, including the uptake and killing of Aspergillus fumigatus (mediated by respiratory burst), produces a large number of cytokines and chemokines, including tumor necrosis factor (TNF), interleukin 1 (IL-1), interleukin 6 (IL-6), and granulocyte monocyte colony stimulating factor (GM-CSF). The deep understanding of the Aspergillus fumigatus The regulation and action mechanism of Dectin-1 in the infection is of great significance to the study of Aspergillus fumigatus and clinical prevention and control. By establishing an in vitro model of Aspergillus fumigatus infection, this study observed the expression changes of Dectin-1 after Aspergillus fumigatus infection, and evaluated Toll like receptor 2 (TLR2) at different levels by siRNA silence technique, and the transcription factor PU.1 to Dectin-1 table. The impact of regulation.
Part one the effect of TLR2 on the expression of Dectin-1 in Aspergillus fumigatus infection
Objective: to establish an in vitro model of human bronchial epithelial cells (HBE cells) infected by Aspergillus fumigatus and observe the changes of Dectin-1, TLR2 expression and the relationship between the two, so as to provide a basis for further research on the synergistic mechanism between different PRRs.
Methods: (1) the spores of Aspergillus fumigatus were infected with HBE cells, and the number of multiplicity of infection (MOI) was 1., respectively, to terminate the infection in 0,6,18,24h, and to extract the total RNA and total protein of the cells. The expression of Dectin-1 was observed by the flow cytometry. Results: after HBE cells infected Aspergillus fumigatus spores 6h, the mRNA expression level of Dectin-1 increased significantly, the difference was statistically significant (P0.05), and the 24h remained at a high level after infection; the expression level of Dectin-1 protein was low in the resting state of HBE cells, and the initial state was lower. After infection, the expression of 6h increased by 11 times (P0.01). The mRNA and protein of TLR2 had a high level of expression in the resting state HBE cells. With the prolongation of the infection time, the protein expression level was higher, but there was no significant difference compared with the resting state expression level (P0.05). (3) silent TLR2 and 1 of Aspergillus fumigatus infection. The expression of Dectin-1 in HBE cells was significantly inhibited at 8h, and the difference was statistically significant (P0.05). Conclusion: in the infection of Aspergillus fumigatus, the expression of Dectin-1 in HBE cells was enhanced, but the expression of TLR2 had no obvious changes; TLR2 has synergistic effect on the expression of Dectin-1, which may be involved in its up-regulated process, but the specific regulatory mechanism is not clear.
Second part of the regulation role of transcription factor PU.1 on Dectin-1 in Aspergillus fumigatus infection
Objective: to construct a THP-1 (human acute leukemia monocyte strain) macrophage Aspergillus fumigatus infection model, and to explore the effect of transcription factor PU.1 on phagocytosis of THP-1 macrophages and the regulation of Dectin-1 transcriptional level in the infection of Aspergillus fumigatus.
Methods: (1) THP-1 macrophages were infected by Aspergillus fumigatus spores, MOI=1. was terminated in 0,12,24h, the total protein was extracted, and the expression of PU.1 protein was detected by Western Blot. PU.1 was silent, THP-1 macrophage infection model was replicated, Western Blot was used to evaluate the Dectin-1 table, and the laser confocal microscopy was used to observe the smoke. Changes in the phagocytosis of Aspergillus spore.
Results: THP-1 macrophages could express the transcription factor PU.1 in the resting state, and the expression increased gradually with the time of infection. The difference was statistically significant (P0.05). After the transcription factor PU.1 was silent, the expression of Dectin-1 in THP-1 macrophages was significantly inhibited when 12h was infected by Aspergillus fumigatus, and the difference was statistically significant (P0.05). After silencing of transcription factor PU.1, THP-1 macrophages decreased the phagocytosis of Aspergillus fumigatus spores during resting stage and swelling stage.
Conclusion: the expression of transcription factor PU.1 is up regulated during Aspergillus fumigatus infection, which affects the phagocytosis of THP-1 macrophages and may participate in the regulation of Dectin-1 expression at the transcriptional level, but the direct connection between the two needs further research.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R519.8

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