本文選題：煙曲霉 + HBE細(xì)胞��； 參考：《第二軍醫(yī)大學(xué)》2013年碩士論文

【摘要】：近年來隨著易感人群的增加，煙曲霉感染的發(fā)病率呈現(xiàn)不斷上升趨勢。即使目前抗真菌藥物研究取得很大進(jìn)步，且臨床診療水平不斷提高，但侵襲性曲霉�。╥nvasiveaspergillosis，IA）的病死率仍高達(dá)50%～90%。煙曲霉感染的研究證明，機(jī)體免疫狀態(tài)是宿主發(fā)病的重要因素。隨著研究的不斷深入，模式識別受體（pattern recognitionreceptors，PRRs）在機(jī)體免疫調(diào)節(jié)中的作用日漸受到重視。人們認(rèn)識到PRRs對病原體相關(guān)分子模式（pathogen-associated molecular patterns，PAMPs）的識別是宿主抗真菌免疫的始動環(huán)節(jié)。深入研究PRRs的作用及其調(diào)節(jié)機(jī)制，為從干預(yù)宿主免疫狀態(tài)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是一種糖基化Ⅱ型跨膜受體，屬于C型植物凝集素家族，主要通過識別β-(1,3)葡聚糖介導(dǎo)宿主抗真菌免疫防御反應(yīng)。在煙曲霉感染中Dectin-1通過識別煙曲霉胞壁的β-(1,3)葡聚糖成份促進(jìn)機(jī)體的保護(hù)性應(yīng)答反應(yīng)，包括對煙曲霉的攝取及殺傷（通過呼吸爆發(fā)介導(dǎo)），產(chǎn)生大量的細(xì)胞因子和趨化因子，包括腫瘤壞死因子（TNF）、白細(xì)胞介素1（IL-1）、白細(xì)胞介素6（IL-6）以及粒細(xì)胞-單核細(xì)胞集落刺激因子（GM-CSF）等。深入了解煙曲霉感染中Dectin-1的調(diào)節(jié)及作用機(jī)制，對研究煙曲霉發(fā)病及臨床防治具有重要意義。本研究通過建立煙曲霉感染的體外模型，觀察Dectin-1在煙曲霉感染后的表達(dá)變化，并通過siRNA沉默技術(shù)在不同水平評價Toll樣受體2（TLR2）、轉(zhuǎn)錄因子PU.1對Dectin-1表達(dá)調(diào)控的影響。 第一部分煙曲霉感染時TLR2對Dectin-1表達(dá)的影響 目的：建立煙曲霉感染人支氣管上皮細(xì)胞（human bronchial epithelial cells，HBE細(xì)胞）的體外模型，觀察Dectin-1、TLR2表達(dá)變化以及二者之間的關(guān)系，為進(jìn)一步研究不同PRRs之間的協(xié)同機(jī)制提供依據(jù)。 方法：①煙曲霉孢子感染HBE細(xì)胞，感染復(fù)數(shù)（multiplicity of infection，MOI）為1。分別在0、6、18、24h終止感染，提取細(xì)胞總RNA、總蛋白，通過Western Blot、PCR方法分別在蛋白、mRNA水平上檢測Dectin-1和TLR2的表達(dá)情況；②沉默TLR2，復(fù)制感染模型，通過Western Blot及流式細(xì)胞技術(shù)觀察Dectin-1的表達(dá)變化。結(jié)果：①HBE細(xì)胞感染煙曲霉孢子6h后，Dectin-1的mRNA表達(dá)水平顯著升高，差異具有統(tǒng)計學(xué)意義（P0.05），在感染后24h仍維持在較高水平；在HBE細(xì)胞靜息狀態(tài)下Dectin-1蛋白基礎(chǔ)表達(dá)水平較低，與初始狀態(tài)相比，感染后6h表達(dá)量增加11倍（P0.01）。②TLR2的mRNA和蛋白在靜息狀態(tài)下的HBE細(xì)胞中即有較高水平表達(dá)，隨著感染時間的延長，其蛋白表達(dá)水平有增高的趨勢，但與靜息狀態(tài)下表達(dá)水平相比差異無統(tǒng)計學(xué)意義（P0.05）。③沉默TLR2，在煙曲霉感染18h時，HBE細(xì)胞中Dectin-1的表達(dá)受到顯著抑制，差異具有統(tǒng)計學(xué)意義（P0.05）。結(jié)論：在煙曲霉感染中，HBE細(xì)胞內(nèi)Dectin-1表達(dá)增強(qiáng)，而TLR2表達(dá)無明顯變化；TLR2對Dectin-1的表達(dá)有協(xié)同作用，可能參與其上調(diào)表達(dá)過程，但具體的調(diào)節(jié)機(jī)制還不清楚。 第二部分煙曲霉感染時轉(zhuǎn)錄因子PU.1對Dectin-1的調(diào)節(jié)作用 目的：構(gòu)建THP-1（人急性白血病單核細(xì)胞株）巨噬細(xì)胞煙曲霉感染模型，探討煙曲霉感染時轉(zhuǎn)錄因子PU.1對THP-1巨噬細(xì)胞吞噬作用的影響和Dectin-1轉(zhuǎn)錄水平的調(diào)節(jié)作用。 方法：①煙曲霉孢子感染THP-1巨噬細(xì)胞，MOI=1。分別在0、12、24h終止感染，提取細(xì)胞總蛋白，通過Western Blot方法檢測PU.1蛋白表達(dá)情況；②沉默PU.1，復(fù)制THP-1巨噬細(xì)胞感染模型，Western Blot評價Dectin-1表達(dá)變化情況，激光共聚焦顯微鏡觀察其對煙曲霉孢子吞噬能力的變化。 結(jié)果：THP-1巨噬細(xì)胞在靜息狀態(tài)下能夠表達(dá)轉(zhuǎn)錄因子PU.1，，隨著感染時間的延長其表達(dá)量逐漸增加，差異具有統(tǒng)計學(xué)意義（P0.05）；轉(zhuǎn)錄因子PU.1沉默后，在煙曲霉感染12h時，THP-1巨噬細(xì)胞中Dectin-1的表達(dá)顯著抑制，差異具有統(tǒng)計學(xué)意義（P0.05）；轉(zhuǎn)錄因子PU.1沉默后，THP-1巨噬細(xì)胞對靜息期、腫脹期煙曲霉孢子的吞噬能力下降。 結(jié)論：在煙曲霉感染時轉(zhuǎn)錄因子PU.1表達(dá)上調(diào)，這影響了THP-1巨噬細(xì)胞的吞噬功能并可能在轉(zhuǎn)錄水平參與調(diào)控Dectin-1的表達(dá)，但是二者之間是否存在直接聯(lián)系還需要進(jìn)一步研究證實(shí)。
[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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R519.8

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