中华危重病急救医学
中華危重病急救醫學
중화위중병급구의학
Chinese Critical Care Medicine
2013年
6期
351-355
,共5页
王宇辉%陈淼%吴艳%钱明江%刘海霞%刘国跃
王宇輝%陳淼%吳豔%錢明江%劉海霞%劉國躍
왕우휘%진묘%오염%전명강%류해하%류국약
血红素加氧酶-1%大鼠%肺泡上皮细胞,Ⅱ型%水通道蛋白-1%凋亡
血紅素加氧酶-1%大鼠%肺泡上皮細胞,Ⅱ型%水通道蛋白-1%凋亡
혈홍소가양매-1%대서%폐포상피세포,Ⅱ형%수통도단백-1%조망
Heme oxygenase-1%Rat%Primary type Ⅱ alveolar epithelial%Aquaporin-1%Apoptosis
目的 探讨血红素加氧酶-1(HO-1)对过氧化氢(H2O2)诱导的氧化损伤大鼠Ⅱ型肺泡上皮细胞(AECⅡ)凋亡及水通道蛋白-1(AQP-1)表达的影响.方法 取雄性SD大鼠肺组织,将分离、纯化的AECⅡ细胞原代培养24 h后分为4组.正常组加入生理盐水；H2O2损伤组加入0.5 mmol/L H2O2处理；HO-1对照组加入1 μmol/L HO-1处理；HO-1保护组同时加入1μmol/L HO-1及0.5 mmol/L H2O2处理,将处理后的细胞继续培养2h.分别于处理前及处理后1、3、6、12h取细胞悬液,采用蛋白质免疫印迹试验(Western blotting)检测AQP-1表达,用流式细胞仪检测细胞凋亡率.结果 H2O2损伤组AQP-1表达水平随时间延长逐渐下降,各时间点明显低于正常组；HO-1对照组和HO-1保护组AQP-1表达水平随时间延长呈上升趋势,各时间点高于其他各组,尤以HO-1保护组明显高于H2O2损伤组(1 h:60.81±5.78比46.21 ±4.81,3 h:63.05 ±9.61比39.32±4.96,6 h:92.59±8.21比36.82±4.32,12 h:86.16±14.84比34.88±2.66,均P＜0.05).正常组和HO-1对照组细胞凋亡率无明显变化；H2O2损伤组细胞凋亡率随时间延长逐渐升高,各时间点均显著高于正常组；HO-1保护组细胞凋亡率于3h内逐渐升高,6h后下降并保持稳定,各时间点均显著低于H2O2损伤组[1 h:(9.04±2.17)％比(15.14±2.47)％,3 h:(12.90±2.04)％比(22.37±4.84)％,6 h:(10.42±1.68)％比(27.83±3.93)％,12 h:(11.97± 1.91)％比(33.63±6.61)％,均P＜0.05].H2O2损伤组AQP-1与细胞凋亡率呈显著负相关(r=-0.723,P＜0.001),并存在回归关系[y=672.548(0.914)x,R2=0.597]；HO-1保护组AQP-1与细胞凋亡率无显著相关性(r=0.210,P=0.193),但存在回归关系[y=e(3.130-59.654/x),R2=0.225].结论 HO-1可上调H2O2氧化损伤AECⅡ细胞的AQP-1表达水平并降低细胞凋亡率；上调AQP-1表达可能是HO-1抗氧化损伤机制之一.
目的 探討血紅素加氧酶-1(HO-1)對過氧化氫(H2O2)誘導的氧化損傷大鼠Ⅱ型肺泡上皮細胞(AECⅡ)凋亡及水通道蛋白-1(AQP-1)錶達的影響.方法 取雄性SD大鼠肺組織,將分離、純化的AECⅡ細胞原代培養24 h後分為4組.正常組加入生理鹽水；H2O2損傷組加入0.5 mmol/L H2O2處理；HO-1對照組加入1 μmol/L HO-1處理；HO-1保護組同時加入1μmol/L HO-1及0.5 mmol/L H2O2處理,將處理後的細胞繼續培養2h.分彆于處理前及處理後1、3、6、12h取細胞懸液,採用蛋白質免疫印跡試驗(Western blotting)檢測AQP-1錶達,用流式細胞儀檢測細胞凋亡率.結果 H2O2損傷組AQP-1錶達水平隨時間延長逐漸下降,各時間點明顯低于正常組；HO-1對照組和HO-1保護組AQP-1錶達水平隨時間延長呈上升趨勢,各時間點高于其他各組,尤以HO-1保護組明顯高于H2O2損傷組(1 h:60.81±5.78比46.21 ±4.81,3 h:63.05 ±9.61比39.32±4.96,6 h:92.59±8.21比36.82±4.32,12 h:86.16±14.84比34.88±2.66,均P＜0.05).正常組和HO-1對照組細胞凋亡率無明顯變化；H2O2損傷組細胞凋亡率隨時間延長逐漸升高,各時間點均顯著高于正常組；HO-1保護組細胞凋亡率于3h內逐漸升高,6h後下降併保持穩定,各時間點均顯著低于H2O2損傷組[1 h:(9.04±2.17)％比(15.14±2.47)％,3 h:(12.90±2.04)％比(22.37±4.84)％,6 h:(10.42±1.68)％比(27.83±3.93)％,12 h:(11.97± 1.91)％比(33.63±6.61)％,均P＜0.05].H2O2損傷組AQP-1與細胞凋亡率呈顯著負相關(r=-0.723,P＜0.001),併存在迴歸關繫[y=672.548(0.914)x,R2=0.597]；HO-1保護組AQP-1與細胞凋亡率無顯著相關性(r=0.210,P=0.193),但存在迴歸關繫[y=e(3.130-59.654/x),R2=0.225].結論 HO-1可上調H2O2氧化損傷AECⅡ細胞的AQP-1錶達水平併降低細胞凋亡率；上調AQP-1錶達可能是HO-1抗氧化損傷機製之一.
목적 탐토혈홍소가양매-1(HO-1)대과양화경(H2O2)유도적양화손상대서Ⅱ형폐포상피세포(AECⅡ)조망급수통도단백-1(AQP-1)표체적영향.방법 취웅성SD대서폐조직,장분리、순화적AECⅡ세포원대배양24 h후분위4조.정상조가입생리염수；H2O2손상조가입0.5 mmol/L H2O2처리；HO-1대조조가입1 μmol/L HO-1처리；HO-1보호조동시가입1μmol/L HO-1급0.5 mmol/L H2O2처리,장처리후적세포계속배양2h.분별우처리전급처리후1、3、6、12h취세포현액,채용단백질면역인적시험(Western blotting)검측AQP-1표체,용류식세포의검측세포조망솔.결과 H2O2손상조AQP-1표체수평수시간연장축점하강,각시간점명현저우정상조；HO-1대조조화HO-1보호조AQP-1표체수평수시간연장정상승추세,각시간점고우기타각조,우이HO-1보호조명현고우H2O2손상조(1 h:60.81±5.78비46.21 ±4.81,3 h:63.05 ±9.61비39.32±4.96,6 h:92.59±8.21비36.82±4.32,12 h:86.16±14.84비34.88±2.66,균P＜0.05).정상조화HO-1대조조세포조망솔무명현변화；H2O2손상조세포조망솔수시간연장축점승고,각시간점균현저고우정상조；HO-1보호조세포조망솔우3h내축점승고,6h후하강병보지은정,각시간점균현저저우H2O2손상조[1 h:(9.04±2.17)％비(15.14±2.47)％,3 h:(12.90±2.04)％비(22.37±4.84)％,6 h:(10.42±1.68)％비(27.83±3.93)％,12 h:(11.97± 1.91)％비(33.63±6.61)％,균P＜0.05].H2O2손상조AQP-1여세포조망솔정현저부상관(r=-0.723,P＜0.001),병존재회귀관계[y=672.548(0.914)x,R2=0.597]；HO-1보호조AQP-1여세포조망솔무현저상관성(r=0.210,P=0.193),단존재회귀관계[y=e(3.130-59.654/x),R2=0.225].결론 HO-1가상조H2O2양화손상AECⅡ세포적AQP-1표체수평병강저세포조망솔；상조AQP-1표체가능시HO-1항양화손상궤제지일.
Objective To explore the effect of heme oxygenase-1 (HO-1) on apoptosis and expression of aquaporin-1 (AQP-1) in primary type Ⅱ alveolar epithelial cells (AEC Ⅱ) in rats with hydrogen peroxide (H2O2) induced oxidative damage.Methods Lung tissue of male Sprague-Dawley (SD) rats was collected.Primary AEC Ⅱ were isolated,purified,and cultured for 24 hours,then they were divided into four groups:① normal group (treated with normal saline); ② H2O2 injury group (treated with H2O2 0.5 mmol/L); ③ HO-1 control group (treated with HO-1 1 μmol/L); ④ HO-1 protection group (treated with HO-1 1 μmol/L and H2O2 0.5 mmol/L).Cells of each group were cultured for 12 hours after various treatment.The cell suspension was collected before and 1,3,6,12 hours after treatment,the expression of AQP-1 was determined by Western blotting and the apoptosis rate was assessed with flow cytometer.Results The expression of AQP-1 in H2O2 injury group was significantly declined with time,and was lower than that in normal group at each time point after treatment.The expression of AQP-1 in HO-1 control group and HO-1 protection group was significantly increased with time,and was higher than that of other groups at each time point after treatment.The expression of AQP-1 in HO-1 protection group was significantly up-regulated compared with that in H2O2 injury group (1 hour:60.81 ± 5.78 vs.46.21 ± 4.81,3 hours:63.05 ± 9.61 vs.39.32 ± 4.96,6 hours:92.59 ± 8.21 vs.36.82 ±4.32,12 hours:86.16 ± 14.84 vs.34.88 ± 2.66,all P＜0.05).No significant difference in apoptosis rate was found between normal group and HO-1 control group.The apoptosis rate in H2O2 injury group was increased with time,and was significantly higher than that of normal group at each time point.The apoptosis rate in HO-1 protection group was gradually increased within 3 hours after treatment,then decreased and remained stable after 6 hours,while it was significantly lower than that of H2O2 injury group at each time point [1 hour:(9.04 ± 2.17)％ vs.(15.14 ± 2.47)％,3 hours:(12.90 ± 2.04)％ vs.(22.37 ± 4.84)％,6 hours:(10.42 ± 1.68)％ vs.(27.83 ± 3.93)％,12 hours:(11.97 ± 1.91)％ vs.(33.63 ± 6.61)％,all P＜0.05].A negative correlation was found between AQP-1 and apoptosis rate in H2O2 injury group (r=-0.723,P＜0.001),and a regression correlation was found [y=672.548(0.914)x,R2=0.597].AQP-1 was not correlated with apoptosis rate in HO-1 protection group (r=0.210,P=0.193),but a regression correlation was found [y=e (3.130-59.654/x),R2=0.225].Conclusions HO-1 could increase the expression of AQP-1 in H2O2 injured AEC Ⅱ of rat,and lower its apoptosis rate.Increase in the expression of AQP-1 may be the underlying mechanism of anti-oxygenation property of HO-1.
