CAJ | 학술논문

目的探讨血红素加氧酶-1 (HO-1)对过氧化氢(H2O2)氧化损伤肺泡Ⅰ型上皮细胞(AEC Ⅰ)凋亡以及线粒体跨膜电位(MTMP)的影响.方法将24只健康雄性SD大鼠腹腔注射麻醉处死后取肺,分离纯化AEC Ⅰ接种于培养板,原代培养24 h后等量分为正常对照组、H2O2损伤组、HO-1保护组、HO-1抑制组4组.采用0.5 mmol/L H2O2刺激AEC Ⅰ 2.5 h建立细胞氧化损伤模型.制模后用0.2μmol/L HO-1或20μmol/L锌原卟啉Ⅸ处理给予保护或抑制.处理2.5h后用荧光显微镜观察细胞的形态学变化；分别于处理0.5、1.0、1.5、2.0、2.5h5个时间点用流式细胞仪检测细胞凋亡率及MTMP去极化比例的变化.结果荧光显微镜下观察.:H2O2损伤组可见大量呈绿色(凋亡早期)、红色(凋亡中晚期)荧光的凋亡细胞；HO-1保护组所见绿色或红色荧光细胞明显减少；HO-1抑制组与H2O2损伤组镜下所见相似.流式细胞仪检测结果显示:与正常对照组比较,H2O2损伤组细胞凋亡率增加,且随H2O2作用时间延长逐渐增加,2.5h达峰值[0.5 h:(30.27±0.74)％比(3.76±0.81)％,2.5 h:(40.46±0.91)％比(22.74±0.60)％,均P＜0.05],MTMP去极化比例明显降低(0.99±0.21比1.91±0.16,P＜0.05)；与H2O2损伤组比较,HO-1保护组AEC Ⅰ细胞凋亡率明显降低[0.5 h:(5.99±0.60)％比(30.27±0.74)％,2.5h:(22.69±1.69)％比(40.46±0.91)％,均P＜0.05],MTMP去极化比例明显升高(2.02±0.12比0.99±0.21,P＜0.05)；与HO-1保护组比较,HO-1抑制组细胞凋亡率明显增加[0.5 h:(30.73±1.08)％比(5.99±0.60)％,2.5h:(41.38±0.57)％比(22.69±1.69)％,均P＜0.05],MTMP去极化比例明显降低(0.98±0.09比2.02±0.12,P＜0.05).结论HO-1可保护AEC Ⅰ细胞完整性,降低细胞凋亡率并且稳定MTMP,对H2O2所致大鼠AEC Ⅰ氧化损伤具有一定的保护作用.
목적 탐토혈홍소가양매-1 (HO-1)대과양화경(H2O2)양화손상폐포Ⅰ형상피세포(AEC Ⅰ)조망이급선립체과막전위(MTMP)적영향.방법 장24지건강웅성SD대서복강주사마취처사후취폐,분리순화AEC Ⅰ접충우배양판,원대배양24 h후등량분위정상대조조、H2O2손상조、HO-1보호조、HO-1억제조4조.채용0.5 mmol/L H2O2자격AEC Ⅰ 2.5 h건립세포양화손상모형.제모후용0.2μmol/L HO-1혹20μmol/L자원계람Ⅸ처리급여보호혹억제.처리2.5h후용형광현미경관찰세포적형태학변화；분별우처리0.5、1.0、1.5、2.0、2.5h5개시간점용류식세포의검측세포조망솔급MTMP거겁화비례적변화.결과 형광현미경하관찰.:H2O2손상조가견대량정록색(조망조기)、홍색(조망중만기)형광적조망세포；HO-1보호조소견록색혹홍색형광세포명현감소；HO-1억제조여H2O2손상조경하소견상사.류식세포의검측결과현시:여정상대조조비교,H2O2손상조세포조망솔증가,차수H2O2작용시간연장축점증가,2.5h체봉치[0.5 h:(30.27±0.74)％비(3.76±0.81)％,2.5 h:(40.46±0.91)％비(22.74±0.60)％,균P＜0.05],MTMP거겁화비례명현강저(0.99±0.21비1.91±0.16,P＜0.05)；여H2O2손상조비교,HO-1보호조AEC Ⅰ세포조망솔명현강저[0.5 h:(5.99±0.60)％비(30.27±0.74)％,2.5h:(22.69±1.69)％비(40.46±0.91)％,균P＜0.05],MTMP거겁화비례명현승고(2.02±0.12비0.99±0.21,P＜0.05)；여HO-1보호조비교,HO-1억제조세포조망솔명현증가[0.5 h:(30.73±1.08)％비(5.99±0.60)％,2.5h:(41.38±0.57)％비(22.69±1.69)％,균P＜0.05],MTMP거겁화비례명현강저(0.98±0.09비2.02±0.12,P＜0.05).결론HO-1가보호AEC Ⅰ세포완정성,강저세포조망솔병차은정MTMP,대H2O2소치대서AEC Ⅰ양화손상구유일정적보호작용.
Objective To understand the role of hemne oxygenase-1 (HO-1) in hydrogen peroxide (H2O2) induced apoptosis and mitochondrial trans-membrane potential (MTMP) change in primary alveolar epithelial cell type Ⅱ (AEC Ⅰ ).Methods Primary AEC Ⅰ collected from healthy Sprague Dawley (SD) rats were cultured for 24 hours,then divided into four groups to be treated with:① saline； ② H2O2 (0.5 mmol/L) ; ③ H2O2 + HO-1 (0.2 mmol/L) ； ④ H2O2 + zinc original porphyrin Ⅸ (HO-1 inhibitor,20 μtmol/L ).The morphology of cells in the cultures was examined by fluorescent microscopy 2.5 hours later,and the number of apoptotic cells/the MTMP determined by flow-cytometry 0.5,1.0,1.5,2.0 and 2.5 hours later.Results Large number of cells in with green (early apoptotic) or red (later apoptotic) fluorescence were observed by microscope in cultures treated with H2O2,and H2O2 + HO-1 inhibitor,but such cells were obviously fewer in HO-1 treated cultures.Compared with saline treated cells,H2O2 treated cells had significantly higher apoptosis rate,that increased with time,reaching peak value 2.5 hours into the treatment [0.5 hour:(30.27±0.74)％ vs.(3.76±0.81)％,2.5 hours:(40.46±0.91)％ vs.(22.74± 0.60)％,both P＜0.05],while the rate of MTMP depolarization was significantly lower (0.99± 0.21 vs.1.91±0.16,P＜0.05) in these cells.Compared with H2O2 treated cells,the apoptosis rate in HO-1 treated cells was significantly lower [0.5 hour:(5.99 ± 0.60)％ vs.(30.27±0.74)％,2.5 hours:(22.69± 1.69)％ vs.(40.46±0.91)％,both P＜0.05],and their rate of MTMP depolarization higher (2.02±0.12 vs.0.99±0.21,P＜0.05).Compared with HO-1 treated cells,HO-1 inhibitor treated cells had significantly higher apoptosis rate which reached peak value 2.5 hours into the treatment [0.5 houri (30.73±1.08)％ vs.(5.99±0.60)％,2.5 hours:(41.38±0.57)％ vs.(22.69±1.69)％,both P＜0.05],while rate of MTMP depolarization in these cells was significantly lower (0.98 ± 0.09 vs.2.02 ± 0.12,P ＜ 0.05).Conclusion HO-1 could maintain the integrity of AEC Ⅰ and stabilize their mitochondria membranepotential,protecting the cells from H2O2 induced damage.