miR-21-5p过表达对高氧性急性肺损伤大鼠AECⅡ早期凋亡的影响  

Effect of overexpression of microRNA-21-5p on early apoptosis of typeⅡalveolar epithelial cells in rats with hyperoxic acute lung injury

作　　者：张伟[1] 徐乐 陈淼[1] 梅鸿[1] 李康[1] 覃松[1] 刘国跃[1] Zhang Wei;Xu Le;Chen Miao;Mei Hong;Li Kang;Qin Song;Liu Guoyue(Department of Intensive Care Unit,Affiliated Hospital of Zunyi Medical College,Zunyi 563003,Guizhou,China;Department of Anesthesiology,Affiliated Hospital of Qingdao University,Qingdao 266003,Shandong,China)

机构地区：[1]遵义医学院附属医院重症医学科,563003 [2]青岛大学附属医院麻醉科,266003

出　　处：《中华危重病急救医学》2019年第8期978-982,共5页Chinese Critical Care Medicine

基　　金：国家自然科学基金(81560019).

摘　　要：目的探讨微小RNA-21-5p(miR-21-5p)过表达对高氧性急性肺损伤(HALI)大鼠Ⅱ型肺泡上皮细胞(AECⅡ)早期凋亡的影响。方法按随机数字表法将SD大鼠分为对照组(CON组)、高氧组(H组)、过表达组(OE组)及空载体组(EV组)4组,每组20只。以吸入90%以上高浓度氧制备HALI动物模型;CON组则吸入空气。OE组及EV组分别经气管导管往肺内滴入miR-21-5p腺相关病毒-?6(AAV-6)过表达载体或空载体,饲养3周后制备HALI模型。各组于制模0、24、48、60h分别取5只动物,检测肺损伤相关指标,如氧合指数(OI)、呼吸指数(RI)、肺湿/干重比值(W/D)、肺组织病理评分;同时采用实时荧光定量聚合酶链反应(RT-qPCR)检测AECⅡ中miR-21-5p表达,用流式细胞仪检测AECⅡ早期凋亡率。结果①肺损伤相关指标:H组OI随时间延长逐渐降低,RI、肺W/D比值及病理评分均随时间延长逐渐增高,24h即与CON组差异有统计学意义〔OI(mmHg,1mmHg=0.133kPa):336.04±5.79比400.22±19.70,RI:0.20±0.02比0.10±0.06,肺W/D比值:5.04±0.09比4.85±0.09,肺组织病理评分(分):0.13±0.01比0.07±0.01,均P<0.05〕,表明持续吸入高浓度氧可成功建立HALI模型。②miR-21-5p表达:H组、OE组及EV组AECⅡ细胞中miR-21-5p表达均随时间延长逐渐增高,且24、48、60h时miR-21-5p表达均显著高于CON组。OE组0、24、48、60h时miR-21-5p表达较H组进一步增高(2-ΔΔCt:3.75±0.11比0.98±0.14,3.98±0.12比1.18±0.13,4.28±0.18比1.49±0.06,4.66±0.12比1.80±0.12,均P<0.05)。③AECⅡ早期凋亡率:H组、OE组及EV组AECⅡ细胞早期凋亡率均随时间延长逐渐升高,且24、48、60h时细胞早期凋亡率均显著高于CON组。而OE组24、48、60h时AECⅡ早期凋亡率较H组显著降低〔(1.22±0.63)%比(2.84±0.59)%,(5.76±0.18)%比(13.10±2.01)%,(29.48±0.48)%比(49.04±1.36)%,均P<0.05〕。④EV组各时间点miR-21-5p表达和AECⅡ早期凋亡率与H组比较差异均无统计学意义。结论miR-21-5p过表达可抑制HALI大鼠AECObjective To investigate the effect of overexpression of microRNA-21-5p(miR-21-5p)on early apoptosis of typeⅡalveolar epithelial cells(AECⅡ)in rats with hyperoxic acute lung injury(HALI).Methods The Sprague-Dawley(SD)rats were randomly divided into four groups:control group(CON group),hyperoxia group(H group),overexpression group(OE group)and empty vector group(EV group),with 20 rats in each group.HALI animal model was made by inhaling high concentration oxygen(oxygen concentration≥90%);CON group was arranged to inhale room air.The miR-21-5p adeno-associated virus-6(AAV-6)overexpression vectors or empty vectors were dripped into the lungs of OE group and EV group through tracheal tube,respectively.The hyperoxia model was prepared after 3 weeks of feeding.At 0,24,48 and 60 hours after making model,5 rats were selected to detect lung injury parameters:oxygenation index(OI),respiratory index(RI),wet/dry ratio(W/D),pathological injury score of lung tissue;real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression of miR-21-5p in AECⅡ,and flow cytometry was used to detect the early apoptotic rate of AECⅡ.Results①The lung injury parameters:in H group,the OI gradually decreased with time,but the RI,lung W/D ratio and pathological score increased gradually with time,the difference between CON group was statistically significant at 24 hours[OI(mmHg,1 mmHg=0.133 kPa):336.04±5.79 vs.400.22±19.70,RI:0.20±0.02 vs.0.10±0.06,lung W/D ratio:5.04±0.09 vs.4.85±0.09,lung tissue pathological score:0.13±0.01 vs.0.07±0.01,all P<0.05].It indicated that HALI model could be successfully established by inhaling high concentration oxygen continuously.②The expression of miR-21-5p:the miR-21-5p was gradually increased in H,OE and EV groups,and the expression of miR-21-5p was significantly higher than that in CON group at 24,48 and 60 hours.Compared with H group,the expression of miR-21-5p was significantly increased further in OE group at 0,24,48 and 60 hours(2-ΔΔCt:3.75±0.11 vs.0.98±

关 键 词：微小RNA-21-5p 高氧性急性肺损伤 肺泡上皮细胞 Ⅱ型 凋亡 

分 类 号：R563[医药卫生—呼吸系统]