CAJ | 학술논문

目的探讨小分子干扰RNA(small interference RNA,SiRNA)抑制高氧暴露下人肺腺癌A549细胞中硫氧还蛋白-2(thioredoxin-2,Trx-2)对还原型烟酰胺腺嘌呤二核苷酸(nicotinamideadenine dinucleotide,NADH)脱氢酶亚单位1(NADH dehydrogenase subunit 1,ND1)及细胞色素C氧化酶Ⅰ(cytochrome C oxidase Ⅰ,COX Ⅰ)表达的影响. 方法体外传代培养A549细胞系,将Trx-2序列特异性的SiRNA转染A549细胞,随机分为无干扰空气组、无干扰高氧组、干扰后空气组与干扰后高氧组.分别于高氧或空气暴露12、24、48 h提取A549细胞总RNA和蛋白.采用逆转录.聚合酶链反应技术测定Trx-2、ND1和COX Ⅰ mRNA水平的表达,Western印迹技术检测Trx-2蛋白的表达. 结果 (1)序列特异性SiRNA可显著抑制Trx-2表达.(2)无干扰高氧组24 h Trx-2mRNA水平为0.7799-4-0.1249,显著高于无干扰空气组(0.4424±0.1140)(P<0.05).干扰后高氧组24 h Trx-2 mRNA水平为0.2774±0.0174、48 h为0.2587±0.0069,均显著低于干扰后空气组和无干扰高氧组(P<O.05).(3)无干扰高氧组24 h ND1 mRNA水平为0.6609±0.0368,显著低于无干扰空气组(0.8898±0.1049)(P<0.05).干扰后高氧组12 h ND1 mRNA水平为0.8848±0.0135,显著高于干扰后空气组(P<0.05).干扰后高氧组48 h ND1 mRNA水平为0.3808±0.0937,均显著低于干扰后空气组和无干扰高氧组(P<0.05).(4)无干扰高氧组12 h COX Ⅰ mRNA水平为1.7313±0.4331、24 h为2.1929±0.6722、48 h为2.0754±0.2584,均显著高于无干扰空气组(P<0.05). 结论 ND1、COX Ⅰ参与高氧肺损伤发生发展过程,Trx-2对高氧肺损伤线粒体起重要的保护作用.
목적 탐토소분자간우RNA(small interference RNA,SiRNA)억제고양폭로하인폐선암A549세포중류양환단백-2(thioredoxin-2,Trx-2)대환원형연선알선표령이핵감산(nicotinamideadenine dinucleotide,NADH)탈경매아단위1(NADH dehydrogenase subunit 1,ND1)급세포색소C양화매Ⅰ(cytochrome C oxidase Ⅰ,COX Ⅰ)표체적영향. 방법 체외전대배양A549세포계,장Trx-2서렬특이성적SiRNA전염A549세포,수궤분위무간우공기조、무간우고양조、간우후공기조여간우후고양조.분별우고양혹공기폭로12、24、48 h제취A549세포총RNA화단백.채용역전록.취합매련반응기술측정Trx-2、ND1화COX Ⅰ mRNA수평적표체,Western인적기술검측Trx-2단백적표체. 결과 (1)서렬특이성SiRNA가현저억제Trx-2표체.(2)무간우고양조24 h Trx-2mRNA수평위0.7799-4-0.1249,현저고우무간우공기조(0.4424±0.1140)(P<0.05).간우후고양조24 h Trx-2 mRNA수평위0.2774±0.0174、48 h위0.2587±0.0069,균현저저우간우후공기조화무간우고양조(P<O.05).(3)무간우고양조24 h ND1 mRNA수평위0.6609±0.0368,현저저우무간우공기조(0.8898±0.1049)(P<0.05).간우후고양조12 h ND1 mRNA수평위0.8848±0.0135,현저고우간우후공기조(P<0.05).간우후고양조48 h ND1 mRNA수평위0.3808±0.0937,균현저저우간우후공기조화무간우고양조(P<0.05).(4)무간우고양조12 h COX Ⅰ mRNA수평위1.7313±0.4331、24 h위2.1929±0.6722、48 h위2.0754±0.2584,균현저고우무간우공기조(P<0.05). 결론 ND1、COX Ⅰ삼여고양폐손상발생발전과정,Trx-2대고양폐손상선립체기중요적보호작용.
Objective To explore the effects of expression of thioredoxin-2(Trx-2) suppressed by small interference RNA(SiRNA) in A549 cells exposed to hyperoxia on expression of nicotinamide adenine dinucleotide(NADH) dehydrogenase subunit 1(ND1)and cytochrome C oxidase Ⅰ(COX Ⅰ). Methods A549 cells were gained by serial subcultivation in vitro and transfered with synthetic Trx-2 sequence-specific SiRNA and then were randomly divided into air group without interference,hyperoxia group without interference,air group after interference,and hyperoxia group after interference.After exposure to oxygen or room air for 12,24 and 48 h,expressions of Trx-2,ND1 and COX Ⅰ mRNA of these cells were detected by reverse transcription-polymerase chain reaction (RT-PCR),and Trx-2 protein was detected by Western blot. Results (1)Sequence-specific SiRNA targeting Trx-2 could significantly down-regulate its expression in A549 cells.(2)Trx-2 mRNA levds in hyperoxia group without interference at 24 h was higher than those in air group without interference(0.7799±0.1249 VS 0.4424±Ⅰ.1140,P<0.05).Th-2 mRNA levels in hyperoxia group after ireedcrence at 24 h and 48 h were 0.2774±0.0174 and 0.2587±0.0069,lower than those in air group after interference and hyperoxia group without interference (P<0.05).(3)ND1 mRNA levels in hyperoxia group without interference at 24 h was 0.6609±0.0368,lower than those in air group without interference(0.8898±0.1049)(P<0.05).ND1 mRNA levels in hyperoxia group after interference at 12 h was 0.8848±0.0135,higher than those in air group after imederence(P<0.05).ND1 mRNA levels in hypemxia group after interference at 48 h was 0.3808±0.0937,lower than those in air group after imerference and hyperoxia group without interference(P<0.05).(4)COXI mRNA levels in hypemxia group without inteference at 12,24 and 48 h were 1.7313±0.4331,2.1929±0.6722 and 2.0754±0.2584,higher than those in air group witheUt interference,respectively (P<0.05). Conclusions ND1 and COXⅠ participate in the development of hyperoxia indUCed lung.injury,and Trx-2 is likely to have protective effect on mitochondria of A549 cells in hyperoxia lung injury.