目的观察高体积分数氧(高氧)暴露后人肺泡Ⅱ型上皮细胞来源的肺腺癌A549细胞中硫氧还蛋白-2(Trx-2)和细胞色素C(Cytc)的动态表达,探讨Trx-2和Cytc表达变化在高氧肺损伤中的作用。方法复苏液氮罐中冻存的人肺腺癌A549细胞系后,加入100g/L胎牛血清的MEM培养基,置于37℃50mL/L二氧化碳(CO2)饱和湿度培养箱中培养。然后体外传代培养A549细胞,待其生长至接近融合状态时,随机分为高氧组和空气组,每组18瓶A549细胞(50mL培养瓶)。高氧组持续暴露于常压高氧中,氧体积分数为900mL/L,CO2体积分数为50mL/L;空气组仍置于50mL/L CO2培养箱中。二组分别于高氧或空气暴露12、24、48h后提取A549细胞总RNA,半定量反转录聚合酶链反应检测Trx-2和Cytc mRNA水平;蛋白印迹法检测A549细胞Trx-2蛋白的表达变化。结果1.与空气组比较,高氧组24h Trx-2 mRNA的表达显著增强(P〈0.05);48h Trx-2 mRNA的表达减弱,二组比较无显著性差异(P〉0.05)。2.与空气组比较,高氧组12h Cytc mRNA的表达显著增强(P〈0.05);24h Cytc mRNA的表达减弱,二组比较无显著性差异(P〉0.05);48h显著减弱(P〈0.05)。3.与空气组比较,高氧组12h Trx-2蛋白的表达显著增强(P〈0.05);24h仍增强,二组比较无显著差异(P〉0.05);48h显著减弱(P〈0.05)。结论高氧暴露可诱导A549细胞中Trx-2和Cytc异常表达,提示Trx-2对高氧肺损伤线粒体可能起重要的保护作用,Cytc在高氧肺损伤细胞凋亡发生发展中起重要作用。
Objective To observe the expression of thioredoxin - 2 ( Trx - 2 ) and cytochrome C ( Cytc ) in human adenocarcinoma of lung A549 cells from human alveolar epithelial cell Ⅱ exposed to hyperoxia, and explore the roles of Trx - 2 and Cytc level changes in hyperoxic lung injury. Methods After the A549 human lung cancer cell lines were resuscitated, adding 100 g/L fetal bovine seium of the medium minimum essential medium(MEM). A549 cells were gained with serial subcuhivation in vitro. As approaching to the condition of confluence in CO2 culture chamber( 37 ℃ ,50 mL/L CO, ), the cells were randomly divided into air group and hyperoxia group, 18 bottles A549 cells in every group. Hyperoxia group was continuously exposed to oxygen (900 mL/L O2 ,50 mL/L CO2 ) , while air group still in specific room air(50 mL/L CO2 ). After exposed to oxygen or room air for 12,24 and 48 hours, RNA in those cells were isolated, and Trx -2 and Cytc mRNA were detected by semiquantitative reverse transcription polymerase chain reaction( RT - PCR ). Western - blotting was used to detect Trx - 2 protein in A549 cells. Results 1. Compared with air group, Trx - 2 mRNA expression was significantly increased after 24 hours in the hyperoxia group( P 〈 0.05 ) ;its expression became weak after 48 hours of hyperoxia group, but there was no significant difference between 2 groups(P 〉 0.05). 2. Compared with air group, Cytc mRNA expression was significantly increased after 12 hours in the hyperoxia group (P 〈 0.05 ) ; its expression became weak after 24 hours of hyperoxia group, but there was no significant difference between 2 groups ( P 〉 0.05 ), its expression was significantly decreased after 48 hours ( P 〈 0.05 ). 3. Compared with air group, Trx - 2 protein expression was significantly increased after 12 hours of hyperoxia group( P 〈 0.05 ) ;the general tendency increased after 24 hours, but there was no significant difference between 2 groups ( P 〉 0.05 ), its expression wa