絲切蛋白（cofilin）在煙曲霉內(nèi)化侵入肺泡上皮細胞過程中的功能研究
發(fā)布時間：2018-08-18 07:44
【摘要】：背景和目的侵襲性肺曲霉病是免疫受損宿主最常見的深部真菌感染,其發(fā)病率、死亡率居高不下,已對人類健康產(chǎn)生嚴重危害,因此對其致病機制的探討具有重要意義。其主要病原菌煙曲霉孢子在被吸入肺泡后可誘發(fā)肺泡上皮細胞肌動蛋白骨架重排而內(nèi)化侵入上皮細胞,這是其逃避宿主巨噬細胞、中性粒細胞等天然免疫細胞攻擊,造成后續(xù)侵襲性肺曲霉病發(fā)生發(fā)展的必要步驟。絲切蛋白(cofilin)是一類肌動蛋白骨架解聚因子,其活性變化在一系列以細胞肌動蛋白骨架重排為基礎(chǔ)的重要生命活動中發(fā)揮調(diào)控作用。研究顯示cofilin在調(diào)節(jié)多種病原微生物侵染宿主細胞過程中發(fā)揮著重要作用,如HIV病毒侵染CD4+T細胞,已成為抗感染藥物研發(fā)的重要靶點之一。本研究旨在研究cofilin在煙曲霉內(nèi)化侵入肺泡上皮細胞過程中的作用及所涉及的信號通路。方法1)應(yīng)用免疫熒光法觀察煙曲霉內(nèi)化侵入肺泡上皮細胞過程中肌動蛋白骨架重排的情況及p-cofilin分布情況。2)應(yīng)用western blot法檢測煙曲霉孢子刺激A549細胞后cofilin/p-cofilin蛋白水平表達。3)應(yīng)用RT-PCR檢測煙曲霉膨脹孢子刺激A549細胞后cofilin轉(zhuǎn)錄水平表達。4)應(yīng)用制霉菌素保護法測定煙曲霉膨脹孢子對不同預(yù)處理的A549的內(nèi)化侵入率。結(jié)果1)煙曲霉膨脹孢子內(nèi)化侵入肺泡上皮細胞伴有肌動蛋白骨架重排,侵入的孢子位于重排的骨架形成的吞噬小體內(nèi)。煙曲霉休眠孢子刺激A549細胞早期對細胞cofilin/p-cofilin蛋白水平表達無影響,一旦孢子膨脹則可引起細胞早期p-cofilin蛋白水平表達動態(tài)變化,呈先升后降,但對總cofilin蛋白水平及轉(zhuǎn)錄水平表達無影響,且增加的p-cofilin定位于肌動蛋白骨架重排形成的吞噬小體上。2)A549細胞cofilin表達下調(diào)、上調(diào)或不能磷酸化均可減弱煙曲霉膨脹孢子的內(nèi)化侵入。A549細胞應(yīng)用LIM激酶抑制劑或過表達slingshot磷酸酶破壞cofilin/p-cofilin狀態(tài)也減弱煙曲霉膨脹孢子的內(nèi)化侵入。3)A549細胞RhoA舌性降低或ROCK活性降低均可通過減少細胞p-cofilin表達而減弱煙曲霉膨脹孢子的內(nèi)化侵入。可溶性β-1,3-葡聚糖模擬煙曲霉膨脹孢子的刺激不能誘發(fā)A549細胞早期cofilin/p-cofilin動態(tài)變化,且應(yīng)用dectin-1特異性抗體封閉細胞表面相應(yīng)受體也不能影響煙曲霉膨脹孢子引起的細胞早期p-cofilin蛋白水平表達變化。結(jié)論煙曲霉膨脹孢子感染肺泡上皮細胞可引起細胞早期cofilin/p-cofilin動態(tài)變化以利孢子有效的內(nèi)化侵入,且是通過RhoA/ROCK/LIMK信號通路調(diào)節(jié),但該過程并不一定由細胞表面的dectin-1受體介導(dǎo)。
[Abstract]:Background and objective invasive pulmonary aspergillosis is the most common deep fungal infection in immunocompromised host. Its morbidity and mortality remain high, which has caused serious harm to human health. Therefore, it is of great significance to explore the pathogenesis of Aspergillus pneumoniae. The main pathogen, Aspergillus fumigatus spores, can induce the rearrangement of actin cytoskeleton in alveolar epithelial cells and internalize the invasion of alveolar epithelial cells after inhalation, which is a way to escape the attack of host macrophages, neutrophils and other innate immune cells. The necessary steps for the subsequent occurrence and development of invasive pulmonary aspergillosis. (cofilin) is a kind of actin cytoskeleton depolymerization factor, and its activity changes play a regulatory role in a series of important life activities based on cytoskeleton rearrangement. Studies have shown that cofilin plays an important role in regulating the infection of host cells by various pathogenic microorganisms, such as HIV virus infecting CD4 T cells, which has become one of the important targets in the development of anti-infective drugs. The purpose of this study was to investigate the role of cofilin in the process of intracellular invasion of Aspergillus fumigatus into alveolar epithelial cells and the signaling pathways involved. Methods 1) immunofluorescence method was used to observe the rearrangement of actin skeleton and the distribution of p-cofilin during the process of invading alveolar epithelial cells by Aspergillus fumigatus. 2) the level of cofilin/p-cofilin protein was detected by western blot method after Aspergillus fumigatus spores stimulated A549 cells. The cofilin transcription level of A549 cells stimulated by Aspergillus fumigatus expansion spores was detected by RT-PCR. The internal invasion rate of A549 cells with different pretreatment was determined by nystatin protection method. Results 1) the expansion spores of Aspergillus fumigatus invaded alveolar epithelial cells with actin skeleton rearrangement, and the invading spores were located in the phagocytic small bodies formed by the rearranged cytoskeleton. Early stimulation by aspergillus fumigatus spores had no effect on the expression of cofilin/p-cofilin protein in A549 cells. Once the spores swelled, the expression of p-cofilin protein in A549 cells increased at first and then decreased. But there was no effect on the expression of total cofilin protein and transcription, and the increased p-cofilin was located on the phagocytosome of actin cytoskeleton rearrangement. 2) the expression of cofilin was down-regulated in A549 cells. Upregulation or no phosphorylation can attenuate the internalization of aspergillus fumigatus expansion spores. A549 cell line uses LIM kinase inhibitor or overexpression of slingshot phosphatase to destroy the cofilin/p-cofilin state and attenuate the internalization of Aspergillus fumigatus expansion spores. 3) RhoA tongue decline in A549 cells Low or low ROCK activity could attenuate the internalization of aspergillus fumigatus expansion spores by reducing the expression of p-cofilin. The stimulation of soluble 尾 -1C 3-glucan to mimic the expansion spores of Aspergillus fumigatus could not induce the early dynamic changes of cofilin/p-cofilin in A549 cells. Moreover, blocking the corresponding receptor on the cell surface with dectin-1 specific antibody could not affect the expression of p-cofilin protein in the early stage of the cells induced by Aspergillus fumigatus expansion spores. Conclusion Aspergillus fumigatus expansion spores infection with alveolar epithelial cells can induce early dynamic changes of cofilin/p-cofilin in order to facilitate the effective internalization of spores, which is regulated by RhoA/ROCK/LIMK signaling pathway, but this process is not necessarily mediated by dectin-1 receptor on the cell surface.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R519.8
[Abstract]:Background and objective invasive pulmonary aspergillosis is the most common deep fungal infection in immunocompromised host. Its morbidity and mortality remain high, which has caused serious harm to human health. Therefore, it is of great significance to explore the pathogenesis of Aspergillus pneumoniae. The main pathogen, Aspergillus fumigatus spores, can induce the rearrangement of actin cytoskeleton in alveolar epithelial cells and internalize the invasion of alveolar epithelial cells after inhalation, which is a way to escape the attack of host macrophages, neutrophils and other innate immune cells. The necessary steps for the subsequent occurrence and development of invasive pulmonary aspergillosis. (cofilin) is a kind of actin cytoskeleton depolymerization factor, and its activity changes play a regulatory role in a series of important life activities based on cytoskeleton rearrangement. Studies have shown that cofilin plays an important role in regulating the infection of host cells by various pathogenic microorganisms, such as HIV virus infecting CD4 T cells, which has become one of the important targets in the development of anti-infective drugs. The purpose of this study was to investigate the role of cofilin in the process of intracellular invasion of Aspergillus fumigatus into alveolar epithelial cells and the signaling pathways involved. Methods 1) immunofluorescence method was used to observe the rearrangement of actin skeleton and the distribution of p-cofilin during the process of invading alveolar epithelial cells by Aspergillus fumigatus. 2) the level of cofilin/p-cofilin protein was detected by western blot method after Aspergillus fumigatus spores stimulated A549 cells. The cofilin transcription level of A549 cells stimulated by Aspergillus fumigatus expansion spores was detected by RT-PCR. The internal invasion rate of A549 cells with different pretreatment was determined by nystatin protection method. Results 1) the expansion spores of Aspergillus fumigatus invaded alveolar epithelial cells with actin skeleton rearrangement, and the invading spores were located in the phagocytic small bodies formed by the rearranged cytoskeleton. Early stimulation by aspergillus fumigatus spores had no effect on the expression of cofilin/p-cofilin protein in A549 cells. Once the spores swelled, the expression of p-cofilin protein in A549 cells increased at first and then decreased. But there was no effect on the expression of total cofilin protein and transcription, and the increased p-cofilin was located on the phagocytosome of actin cytoskeleton rearrangement. 2) the expression of cofilin was down-regulated in A549 cells. Upregulation or no phosphorylation can attenuate the internalization of aspergillus fumigatus expansion spores. A549 cell line uses LIM kinase inhibitor or overexpression of slingshot phosphatase to destroy the cofilin/p-cofilin state and attenuate the internalization of Aspergillus fumigatus expansion spores. 3) RhoA tongue decline in A549 cells Low or low ROCK activity could attenuate the internalization of aspergillus fumigatus expansion spores by reducing the expression of p-cofilin. The stimulation of soluble 尾 -1C 3-glucan to mimic the expansion spores of Aspergillus fumigatus could not induce the early dynamic changes of cofilin/p-cofilin in A549 cells. Moreover, blocking the corresponding receptor on the cell surface with dectin-1 specific antibody could not affect the expression of p-cofilin protein in the early stage of the cells induced by Aspergillus fumigatus expansion spores. Conclusion Aspergillus fumigatus expansion spores infection with alveolar epithelial cells can induce early dynamic changes of cofilin/p-cofilin in order to facilitate the effective internalization of spores, which is regulated by RhoA/ROCK/LIMK signaling pathway, but this process is not necessarily mediated by dectin-1 receptor on the cell surface.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R519.8
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