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目的研究对氧磷酶1(PON1)在铜绿假单胞菌绿脓素生成及绿脓素所致细胞损伤中的作用,探讨其在防治肺部铜绿假单胞菌感染方面的可能作用。方法①以293细胞为表达细胞,分为pcDNA 3.1(+)-PON1转染组、pcD-NA3.1(+)-EGFP转染组及生理盐水组,转染细胞后,将细胞蛋白提取物或生理盐水混入铜绿假单胞菌培养物中,过夜培养后检测培养物中密度感知系统信号分子——酰基高丝氨酸内酯(N-acylhomoserine lactone,AHL)及绿脓素水平。②以A549细胞为效应细胞,分为转染组、未转染组及生理盐水组。转染组采用pcDNA3.1(+)-PON1转染A549细胞后并经G418筛选的阳性克隆细胞,未转染组采用普通A549细胞,培养液中加入绿脓素(30μg/ml),生理盐水组为培养液中加入生理盐水的普通A549细胞,观察细胞内活性氧(ROS)、丙二醛(MDA)水平及超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性。结果①pcDNA3.1(+)-PON1转染组铜绿假单胞菌培养物中AHL信号分子水平和绿脓素水平较生理盐水组和pcDNA3.1(+)-EGFP转染组均明显降低;②绿脓素作用后,未转染组细胞内氧化应激水平较生理盐水组明显升高,而转染组较未转染组有所下降。结论PON1不仅能够减少铜绿假单胞菌绿脓素的生成,而且可以减轻绿脓素所致的细胞氧化损伤,可能具有防治铜绿假单胞菌感染的潜在价值。
Objective To investigate the role of PON1 in the production of pyocyanin in Pseudomonas aeruginosa and the cellular injury induced by pyocyanin and to explore the possible role of PON1 in the prevention and treatment of pulmonary Pseudomonas aeruginosa infection. Methods ① 293 cells were divided into pcDNA3.1 (+) - PON1 transfection group, pcD-NA3.1 (+) - EGFP transfection group and normal saline group. After transfection, the cell protein extracts Or saline into Pseudomonas aeruginosa cultures. After overnight culture, the density-sensing signaling molecules such as N-acylhomoserine lactone (AHL) and pyocyanin in the culture were assayed. ② A549 cells as effector cells were divided into transfection group, untransfected group and saline group. The transfected A549 cells transfected with pcDNA3.1 (+) - PON1 cells and positive clonal cells screened by G418, untransfected group using ordinary A549 cells, the culture solution was added pyocyanin (30μg / ml), saline Groups were normal A549 cells with physiological saline added to the culture medium. The activity of intracellular reactive oxygen species (ROS), malondialdehyde (MDA) and the activities of superoxide dismutase (SOD) and catalase (CAT) were observed. Results ① The level of AHL signal and the level of pyocyanin in the Pseudomonas aeruginosa culture of pcDNA3.1 (+) - PON1 transfection group were significantly lower than those in the normal saline group and pcDNA3.1 (+) - EGFP transfection group. After pyocyanin treatment, the level of intracellular oxidative stress in untransfected cells was significantly higher than that in saline group, but decreased in transfection group compared with untransfected cells. Conclusion PON1 can not only reduce the production of Pseudomonas aeruginosa pyocyanin, but also reduce the cellular oxidative damage caused by pyocyanin, which may have the potential value of preventing Pseudomonas aeruginosa infection.