| [Objective ] MP is one of the smallest self-replicating organisms that are capable of cell-free existence. MP possesses an extremely small genome and has no ability to synthesize cell walls and other essential elements for living. These characters determine the specific parasitic way, and MP is also considered as parasite adhering to host cells.MP is a human pathogen which is able to cause community acquired respiratory infections in adults and children. It is also one of the important pathogen of the tracheobronchitis and atypical pneumoniae. MP can not only cause respiratory tract infections but also trigger asthma. And it can also cause multi-system and multi-organ extrapulmonary complications.However, the pathogenesis of MP infection is not very clear so far. Now there are three hypothesis of cytoadherence to respiratory epithelium, immunological mechanism and direct invasion of MP.Recently the role of cytokines in MP infection receives more and more attention. Now the elevation of IL-2, 4, 5, 6, 10, 12, 18 and INF has already been detected in bronchoalveolar lavages (BALs), bloods, and lungs in MP-infected patients. And previous researches indicated that the infection of MP to human lung epithelium cell A549 can induce the change of proteins in cells and the secretion of cytokines such as IL-1, 8, TNF. IL-2, 6, 8, 10, 18 have relation with the severity of diseases caused by MP, IL-4, 5 probably participate in anaphylactic diseases caused by MP, and MP could participate in airway remolding by hasten the precipitation of collagen proteinthrough TGF-β1.Although adherence and interaction of MP to respiratory tract epithelium cells are a critical step in the early stage of infection, we know little about the reaction of the respiratory tract epithelium cells after MP infection at present. And today's researches are mainly based on the analysis and identification of one or a few known proteins. Such researches lack integrity and systematization and are unable to analyze unknown proteins. These deficiencies inhibit the further study about mechanisms of infection.Upon the advent of postgenomic era, proteomics become the hot spot in biological science. It studies the proteome, analyzes the dynamic changes of protein composition, expression and modification in cells, discovers interaction and connection among proteins, and studies the law of changes in the construction and regulation of proteins from the integral angle. 2-D electrophoresis and matrix-assisted laser desorption ionization-time of flight mass spectrometric (MALDI-TOF-MS) are the most classical technologies used in proteomics. Nowadays proteomics has already been widely used in study on pathogenesis of diseases.This study used proteomics to isolate and identify the proteins in respiratory tract epithelium cells after MP infection from the integral angle for the first time. Comparison between protein expression in the normal and MP infected A549 cells can contribute to understanding the role of respiratory tract epithelium cells in the MP infection and provide theoretical basis and further study direction for elucidating the pathogenesis of MP infection.[ Methods] In vitro experiment, we chose A549 cells and MP strain, and determined approximate density of MP. 12 hours after A549 cells were infected by MP, we collected A549 cells and extracted intracellular proteins. At the same time, the control was also established. we quantitated extracted proteins through Bradford method, then they was subjected to isoelectric focusing (IEF) on an IPG strip (pH 4-7 NL, 17cm). The second separation was carried out on 12.5% SDS slab gels and silver-staining was performed. The digitized images then were analyzed with PDQuest software in order to establish the differential expression profiles betweenthe MP-treated and the control. Differential expressed protein spots were cut from the gels and subjected to in-gel digestion with trypsin. Peptide mixtures were analyzed with a voyager-DE STR MALDI-TOF mass spectrometer using a delayed ion extraction and ion mirror reflector mass spectrometer (Applied Biosystems). The primary data acquired in MS were preliminarily identified using the MASCOT software and the database (NCBInr).[Results] We did not discover conspicuous morphological changes between the MP-treated and the control under the contrast phase microscope. In 2-DE maps, we detected average 968 spots in the control groups, and 976 spots in the MP-treated groups. Through PDQuest software analysis the new or disappeared spots were not detected in the MP-treated, but contrast to the control 26 spots in the MP-treated up-regulated and 3 spots down-regulated significantly. Using MALDI-TOF-MS technology and bioinformation, we preliminarily identified 11 protein spots up-regulated in the MP-treated, they are vimentin, and Glucose-6-phosphate 1-dehydrogenase (G6PD) etc. [Conclusions]1, MP infection may affect protein expression in A549 cells;2, Proteomic approach can be applied in study on pathogenesis of pathogen.
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