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目的观察NADPH氧化酶抑制剂二苯基碘(DPI)和夹竹桃麻素(apocynin)对NADPH氧化酶亚基p47phox介导的活性氧(ROS)产生的影响,探讨细胞在高氧条件下,p47phox介导ROS产生的机制。方法 32周以下早产儿,尚未吸氧前取外周血2 m L,分离纯化外周血单个核细胞(PBMC)将所得细胞分为对照组、高氧组、高氧联合DPI处理组、高氧联合apocynin处理组进行培养。对照组置于37℃、50 m L/L的CO2培养箱中,高氧及相应处理组置于950 m L/L的O2与50 m L/L的CO2混合气体中培养48 h。采用Mitosox Red标记结合激光共聚焦显微镜检测PBMC内ROS的生成量、硫代巴比妥酸比色法检测培养液丙二醛含量、免疫荧光检测p47phox在细胞内的定位及移位率、Western blot法检测p47phox的蛋白水平。结果与高氧组相比,其余三组ROS和丙二醛明显减少,p47phox移位率与含量也显著降低;与对照组相比,高氧联合DPI处理组及高氧联合apocynin处理组ROS、丙二醛、p47phox移位率与含量并无显著差异。结论 DPI和apocynin能通过降低p47phox移位与含量来减少高氧诱导的ROS升高。
Objective To investigate the effects of NADPH oxidase inhibitor diphenyliodide (DPI) and apocynin on the production of reactive oxygen species (ROS) mediated by the NADPH oxidase subunit p47phox, p47phox mediates the mechanism of ROS production. Methods Peripheral blood mononuclear cells (PBMCs) were isolated and purified from peripheral blood of 32-week-old preterm infants without inhaled oxygen before inhalation. The cells were divided into control group, hyperoxia group, hyperoxia group and DPI-treated group. Apocynin treatment groups were cultured. The control group was placed in a CO2 incubator at 37 ℃ and 50 m L / L. The hyperoxia group and the corresponding treatment group were cultured in a mixed gas of 950 m L / L of O2 and 50 m L / L of CO2 for 48 h. Mitosox Red labeling combined with laser confocal microscopy was used to detect the production of ROS in PBMC. The content of malondialdehyde in culture medium was detected by thiobarbituric acid colorimetric assay. The localization and translocation rate of p47phox in cells were detected by immunofluorescence. Method to detect p47phox protein levels. Results Compared with hyperoxia group, ROS and malondialdehyde (MDA) and malondialdehyde (MDA) in the remaining three groups were significantly decreased and p47phox translocation rate and content were significantly decreased. Compared with the control group, the ROS in the hyperoxia group treated with hyperoxia combined with apocynin treatment, Malondialdehyde, p47phox shift rate and content were not significantly different. Conclusions DPI and apocynin can reduce hyperoxia-induced increase of ROS by decreasing p47phox shift and content.