摘要目的 观察微小RNA-21(miR-21)抑制剂对高氧性急性肺损伤(HALI)大鼠Ⅱ型肺泡上皮细胞(AECⅡ)凋亡的影响.方法 将80只SD大鼠按随机数字表法分为空气对照组、高氧损伤组、空病毒对照组(经鼻腔滴入200μL慢病毒液)及miR-21抑制剂预处理组(经鼻腔滴入200 μL含miR-21抑制剂的慢病毒液),每组20只;各组大鼠给予相应处理后立即置于氧浓度＞90％的高氧箱中饲养以建立HALI模型;空气对照组正常饲养且不予任何处理.各组分别于高氧暴露0、24、48、72 h随机取10只大鼠,观察双肺组织大体变化,光镜下观察病理学改变.另于48 h取10只大鼠肺组织,采用实时荧光定量反转录-聚合酶链反应(RT-qPCR)检测miR-21表达,采用蛋白质免疫印迹试验(Western Bolt)检测天冬氨酸特异性半胱氨酸蛋白酶3(caspase-3)蛋白表达,采用原位末端缺刻标记试验(TUNEL)检测AECⅡ凋亡情况.结果 ①空气对照组各时间点肺组织无异常表现.随高氧暴露时间延长,高氧损伤组肺组织损伤逐渐加重,72 h肺组织呈暗红色,多处斑片状出血,肺组织结构紊乱,出现肺泡壁断裂,肺泡间隔明显水肿、增宽,大量炎性细胞浸润,肺泡腔内出现水肿液.miR-21抑制剂预处理组肺组织损伤程度较高氧损伤组进一步加重;而空病毒对照组无明显变化.②与空气对照组比较,高氧损伤组肺组织miR-21表达明显下调(2-△△Ct:0.021±0.005比0.037±0.006),caspase-3蛋白表达明显上调(A值:0.423±0.081比0.123±0.023,均P＜ 0.05).给予miR-21抑制剂预处理后,miR-21表达进一步下调(2-△△Ct:0.014±0.003比0.021 ±0.005),caspase-3蛋白表达进一步上调(A值:0.691±0.085比0.423±0.081,均P＜ 0.05);空病毒对照组miR-21(2-△△Ct:0.025±0.007比0.021±0.005)和caspase-3表达(A值:0.475±0.062比0.423±0.081)与高氧损伤组差异无统计学意义(均P＞0.05).③与空气对照组比较,高氧损伤组凋亡细胞增多.给予miR-21抑制剂预处理后,凋亡细胞较高氧损伤组进一步增多;而空病毒对照组则无明显变化.结论 抑制体内miR-21表达可以加重HALI大鼠肺组织损伤,增加AECⅡ凋亡.

abstractsObjective To observe the effects of microRNA-21 (miR-21) inhibitor on apoptosis of type Ⅱalveolar epithelial cells (AEC Ⅱ) in rats with hyperoxia-induced acute lung injury (HALI).Methods Eighty Sprague-Dawley (SD) rats were divided into air-control group,hyperoxia injury group,empty-virus control group (200 μL solution with lentivirus was dropped into the nasal) and miR-21 inhibitor pretreatment group (200 μL solution with lentivirus contained miR-21 inhibitor was dropped through the nasal) by random number table.After treatment,the rats in all groups were fed in the hyperoxia incubator with oxygen concentration exceeding 90％ for production of HALI model,and the rats in air-control group were fed normally without any treatment.Ten rats were selected at 0,24,48 and 72 hours after exposure in hyperoxia environment respectively,and the general changes of lung tissues were observed in light microscope.The right lung tissues were harvested to observe the pathological changes under light microscopy.The left lung tissues of other 10 rats in each group were harvested at 48 hours after execution,the miR-21 expression was determined by real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR),the protein expression of cysteinyl aspartate-specific proteinase-3 (caspase-3) was determined by Western Bolt,and apoptosis of AEC Ⅱ was detected by TdT-mediated dUTP nick end labeling (TUNEL).Results ① No abnormal appearance in lung tissues was observed at all time points in the air-control group.In hyperoxia injury group,the lung injury would be more severe if the exposure time was longer,and lung tissues turned dark red after exposure for 72 hours,with patchy hemorrhage in several places;the structure of lung tissues was disordered,the alveolar wall was broken,the alveolar septum was significantly edematous and broadened,and there was plenty of inflammatory cell infiltration and edema fluid appeared inside the alveolar space.In miR-21 inhibitor pretreatment group,the degree of lung tissue injury was more severe than that of the hyperoxia injury group,and there was no significant change in empty-virus control group.(②) Compared with air-control group,miR-21 expression of the hyperoxia injury group was significantly decreased (2-△△Ct:0.021 ± 0.005 vs.0.037 ± 0.006),and the protein expression of caspase-3 was significantly increased (A value:0.423±0.081 vs.0.123±0.023,both P ＜ 0.05).After pretreatment with miR-21 inhibitor,the expression of miR-21 was further decreased (2-△△Ct:0.014±0.003 vs.0.021 ±0.005),while the protein expression of caspase-3was further increased (A value:0.691 ±0.085 vs.0.423 ±0.081,both P ＜ 0.05).There were no statistically significant differences in the expression of miR-21 (2-△ △ct:0.025 ± 0.007 vs.0.021 ± 0.005) and caspase-3 (A value:0.475 ± 0.062vs.0.423 ±0.081) between empty-virus control group and hyperoxia injury group (both P ＞ 0.05).(③) Compared with air-control group,the apoptosis cells in hyperoxia injury group were increased,which was further increased after pretreatment of miR-21 inhibitor,but no changes were found in empty-virus control group.Conclusion Inhibition of miR-21 expression in vivo could aggravate the injury of lung tissue in HALI rats,and increase the apoptosis of AEC Ⅱ.