丝切蛋白（cofilin）在烟曲霉内化侵入肺泡上皮细胞过程中的功能研究
发布时间：2018-08-18 07:44
【摘要】：背景和目的侵袭性肺曲霉病是免疫受损宿主最常见的深部真菌感染,其发病率、死亡率居高不下,已对人类健康产生严重危害,因此对其致病机制的探讨具有重要意义。其主要病原菌烟曲霉孢子在被吸入肺泡后可诱发肺泡上皮细胞肌动蛋白骨架重排而内化侵入上皮细胞,这是其逃避宿主巨噬细胞、中性粒细胞等天然免疫细胞攻击,造成后续侵袭性肺曲霉病发生发展的必要步骤。丝切蛋白(cofilin)是一类肌动蛋白骨架解聚因子,其活性变化在一系列以细胞肌动蛋白骨架重排为基础的重要生命活动中发挥调控作用。研究显示cofilin在调节多种病原微生物侵染宿主细胞过程中发挥着重要作用,如HIV病毒侵染CD4+T细胞,已成为抗感染药物研发的重要靶点之一。本研究旨在研究cofilin在烟曲霉内化侵入肺泡上皮细胞过程中的作用及所涉及的信号通路。方法1)应用免疫荧光法观察烟曲霉内化侵入肺泡上皮细胞过程中肌动蛋白骨架重排的情况及p-cofilin分布情况。2)应用western blot法检测烟曲霉孢子刺激A549细胞后cofilin/p-cofilin蛋白水平表达。3)应用RT-PCR检测烟曲霉膨胀孢子刺激A549细胞后cofilin转录水平表达。4)应用制霉菌素保护法测定烟曲霉膨胀孢子对不同预处理的A549的内化侵入率。结果1)烟曲霉膨胀孢子内化侵入肺泡上皮细胞伴有肌动蛋白骨架重排,侵入的孢子位于重排的骨架形成的吞噬小体内。烟曲霉休眠孢子刺激A549细胞早期对细胞cofilin/p-cofilin蛋白水平表达无影响,一旦孢子膨胀则可引起细胞早期p-cofilin蛋白水平表达动态变化,呈先升后降,但对总cofilin蛋白水平及转录水平表达无影响,且增加的p-cofilin定位于肌动蛋白骨架重排形成的吞噬小体上。2)A549细胞cofilin表达下调、上调或不能磷酸化均可减弱烟曲霉膨胀孢子的内化侵入。A549细胞应用LIM激酶抑制剂或过表达slingshot磷酸酶破坏cofilin/p-cofilin状态也减弱烟曲霉膨胀孢子的内化侵入。3)A549细胞RhoA舌性降低或ROCK活性降低均可通过减少细胞p-cofilin表达而减弱烟曲霉膨胀孢子的内化侵入。可溶性β-1,3-葡聚糖模拟烟曲霉膨胀孢子的刺激不能诱发A549细胞早期cofilin/p-cofilin动态变化,且应用dectin-1特异性抗体封闭细胞表面相应受体也不能影响烟曲霉膨胀孢子引起的细胞早期p-cofilin蛋白水平表达变化。结论烟曲霉膨胀孢子感染肺泡上皮细胞可引起细胞早期cofilin/p-cofilin动态变化以利孢子有效的内化侵入,且是通过RhoA/ROCK/LIMK信号通路调节,但该过程并不一定由细胞表面的dectin-1受体介导。
[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.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：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.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R519.8
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