硫氧还蛋白-2和细胞色素C在高氧暴露下A549细胞中的表达和意义

Expression of Thioredoxin-2 and Cytochrome C in A549 Cells Exposed to Hyperoxia and Their Significance

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100 g/L胎牛血清的MEM培养基,置于37℃50 mL/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(50 mL培养瓶)。高氧组持续暴露于常压高氧中,氧体积分数为900 mL/L,CO2体积分数为50 mL/L;空气组仍置于50 mL/LCO2培养箱中。二组分别于高氧或空气暴露12、24、48 h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24 h Trx-2 mRNA的表达显著增强(P<0.05);48 h 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12 h Cytc mRNA的表达显著增强(P<0.05);24 h Cytc mRNA的表达减弱,二组比较无显著性差异(P>0.05);48 h显著减弱(P<0.05)。3.与空气组比较,高氧组12 h Trx-2蛋白的表达显著增强(P<0.05);24 h仍增强,二组比较无显著差异(P>0.05);48 h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 was significantly decreased after 48 hours （ P 〈 0.05 ）. Conclusions Exposed to hyperoxia can significantly change the expression of Trx -2 and Cytc in A549 cells, which indicates that Trx -2 likely protects mitochondria of A549 cells in hyperoxic lung injury, and Cytc plays an important role in the process of cell apoptosis in hyperoxic lung injury.