目的 观察严重烧伤早期大鼠心肌组织中微小RNA-126与血清心肌肌钙蛋白Ⅰ(cTnⅠ)的表达变化以及两者的相关性,并在细胞水平验证微小RNA-126与心肌损害的关系.方法 (1)将48只SD大鼠按照随机数字表法分为假伤组8只(致假伤,不予补液)和烧伤组40只(背部造成30％ TBSAⅢ度烫伤,以下称烧伤,伤后经腹腔注射乳酸林格液).假伤组伤后1h收集腹主动脉血,之后处死大鼠取左心室组织;伤后3、6、12、24、48 h,烧伤组分别取8只大鼠采集腹主动脉血,之后处死大鼠取左心室组织.实时荧光定量RT-PCR法检测心肌组织中微小RNA-126的表达水平,ELISA法检测血清cTnⅠ水平.(2)将大鼠心肌细胞株H9C2细胞按随机数字表法分为正常对照组(常规培养)、刺激组、阴性转染+刺激组、转染+刺激组.刺激组将细胞置于含体积分数10％烧伤大鼠血清的DMEM培养液中行缺氧处理24 h;阴性转染+刺激组、转染+刺激组分别用微小RNA模拟物阴性对照、微小RNA-126模拟物转染细胞24 h后,同刺激组处理.其中烧伤大鼠血清来源于实验(1)伤后6h留取血清.实时荧光定量RT-PCR法检测心肌细胞中微小RNA-126表达(样本数为3),用细胞计数试剂盒8检测心肌细胞活力(样本数为4,数据以吸光度值表示),流式细胞仪检测心肌细胞凋亡率(样本数为3).对数据行单因素方差分析、LSD-t检验,对大鼠心肌组织微小RNA-126表达与血清cTnⅠ水平行直线相关分析.结果 (1)与假伤组伤后1h比较,烧伤组伤后各时相点大鼠心肌组织微小RNA-126表达水平均明显降低(t值为5.68 ～9.79,P值均小于0.01),其中伤后24 h达最低值(0.40±0.08);与假伤组伤后1h比较,烧伤组伤后各时相点大鼠血清cTnⅠ水平均明显升高(t值为6.68 ～ 12.79,P值均小于0.01),其中伤后12 h达峰值[(1 035±177)pg/mL].烧伤组大鼠各时相点心肌组织微小RNA-126表达水平与血清cTnⅠ水平呈明显负相关(r=-0.797,P＜0.001).(2)与正常对照组比较,刺激组、转染+刺激组心肌细胞微小RNA-126表达分别明显下降、升高(t值分别为4.57、5.73,P＜0.05或P＜0.01),心肌细胞活力均明显下降(t值分别为14.88、6.48,P值均小于0.01),心肌细胞凋亡率均显著升高(t值分别为13.82、6.96,P值均小于0.01);与阴性转染+刺激组比较,转染+刺激组心肌细胞微小RNA-126表达明显升高(t=6.77,P＜0.01),心肌细胞活力明显升高(t=8.23,P＜0.001),心肌细胞凋亡率明显降低(t=6.14,P＜0.001).结论 大鼠严重烧伤早期心肌组织微小RNA-126表达下降,其可能参与烧伤早期心肌损害的调控并发挥保护作用.
目的 觀察嚴重燒傷早期大鼠心肌組織中微小RNA-126與血清心肌肌鈣蛋白Ⅰ(cTnⅠ)的錶達變化以及兩者的相關性,併在細胞水平驗證微小RNA-126與心肌損害的關繫.方法 (1)將48隻SD大鼠按照隨機數字錶法分為假傷組8隻(緻假傷,不予補液)和燒傷組40隻(揹部造成30％ TBSAⅢ度燙傷,以下稱燒傷,傷後經腹腔註射乳痠林格液).假傷組傷後1h收集腹主動脈血,之後處死大鼠取左心室組織;傷後3、6、12、24、48 h,燒傷組分彆取8隻大鼠採集腹主動脈血,之後處死大鼠取左心室組織.實時熒光定量RT-PCR法檢測心肌組織中微小RNA-126的錶達水平,ELISA法檢測血清cTnⅠ水平.(2)將大鼠心肌細胞株H9C2細胞按隨機數字錶法分為正常對照組(常規培養)、刺激組、陰性轉染+刺激組、轉染+刺激組.刺激組將細胞置于含體積分數10％燒傷大鼠血清的DMEM培養液中行缺氧處理24 h;陰性轉染+刺激組、轉染+刺激組分彆用微小RNA模擬物陰性對照、微小RNA-126模擬物轉染細胞24 h後,同刺激組處理.其中燒傷大鼠血清來源于實驗(1)傷後6h留取血清.實時熒光定量RT-PCR法檢測心肌細胞中微小RNA-126錶達(樣本數為3),用細胞計數試劑盒8檢測心肌細胞活力(樣本數為4,數據以吸光度值錶示),流式細胞儀檢測心肌細胞凋亡率(樣本數為3).對數據行單因素方差分析、LSD-t檢驗,對大鼠心肌組織微小RNA-126錶達與血清cTnⅠ水平行直線相關分析.結果 (1)與假傷組傷後1h比較,燒傷組傷後各時相點大鼠心肌組織微小RNA-126錶達水平均明顯降低(t值為5.68 ～9.79,P值均小于0.01),其中傷後24 h達最低值(0.40±0.08);與假傷組傷後1h比較,燒傷組傷後各時相點大鼠血清cTnⅠ水平均明顯升高(t值為6.68 ～ 12.79,P值均小于0.01),其中傷後12 h達峰值[(1 035±177)pg/mL].燒傷組大鼠各時相點心肌組織微小RNA-126錶達水平與血清cTnⅠ水平呈明顯負相關(r=-0.797,P＜0.001).(2)與正常對照組比較,刺激組、轉染+刺激組心肌細胞微小RNA-126錶達分彆明顯下降、升高(t值分彆為4.57、5.73,P＜0.05或P＜0.01),心肌細胞活力均明顯下降(t值分彆為14.88、6.48,P值均小于0.01),心肌細胞凋亡率均顯著升高(t值分彆為13.82、6.96,P值均小于0.01);與陰性轉染+刺激組比較,轉染+刺激組心肌細胞微小RNA-126錶達明顯升高(t=6.77,P＜0.01),心肌細胞活力明顯升高(t=8.23,P＜0.001),心肌細胞凋亡率明顯降低(t=6.14,P＜0.001).結論 大鼠嚴重燒傷早期心肌組織微小RNA-126錶達下降,其可能參與燒傷早期心肌損害的調控併髮揮保護作用.
목적 관찰엄중소상조기대서심기조직중미소RNA-126여혈청심기기개단백Ⅰ(cTnⅠ)적표체변화이급량자적상관성,병재세포수평험증미소RNA-126여심기손해적관계.방법 (1)장48지SD대서안조수궤수자표법분위가상조8지(치가상,불여보액)화소상조40지(배부조성30％ TBSAⅢ도탕상,이하칭소상,상후경복강주사유산림격액).가상조상후1h수집복주동맥혈,지후처사대서취좌심실조직;상후3、6、12、24、48 h,소상조분별취8지대서채집복주동맥혈,지후처사대서취좌심실조직.실시형광정량RT-PCR법검측심기조직중미소RNA-126적표체수평,ELISA법검측혈청cTnⅠ수평.(2)장대서심기세포주H9C2세포안수궤수자표법분위정상대조조(상규배양)、자격조、음성전염+자격조、전염+자격조.자격조장세포치우함체적분수10％소상대서혈청적DMEM배양액중행결양처리24 h;음성전염+자격조、전염+자격조분별용미소RNA모의물음성대조、미소RNA-126모의물전염세포24 h후,동자격조처리.기중소상대서혈청래원우실험(1)상후6h류취혈청.실시형광정량RT-PCR법검측심기세포중미소RNA-126표체(양본수위3),용세포계수시제합8검측심기세포활력(양본수위4,수거이흡광도치표시),류식세포의검측심기세포조망솔(양본수위3).대수거행단인소방차분석、LSD-t검험,대대서심기조직미소RNA-126표체여혈청cTnⅠ수평행직선상관분석.결과 (1)여가상조상후1h비교,소상조상후각시상점대서심기조직미소RNA-126표체수평균명현강저(t치위5.68 ～9.79,P치균소우0.01),기중상후24 h체최저치(0.40±0.08);여가상조상후1h비교,소상조상후각시상점대서혈청cTnⅠ수평균명현승고(t치위6.68 ～ 12.79,P치균소우0.01),기중상후12 h체봉치[(1 035±177)pg/mL].소상조대서각시상점심기조직미소RNA-126표체수평여혈청cTnⅠ수평정명현부상관(r=-0.797,P＜0.001).(2)여정상대조조비교,자격조、전염+자격조심기세포미소RNA-126표체분별명현하강、승고(t치분별위4.57、5.73,P＜0.05혹P＜0.01),심기세포활력균명현하강(t치분별위14.88、6.48,P치균소우0.01),심기세포조망솔균현저승고(t치분별위13.82、6.96,P치균소우0.01);여음성전염+자격조비교,전염+자격조심기세포미소RNA-126표체명현승고(t=6.77,P＜0.01),심기세포활력명현승고(t=8.23,P＜0.001),심기세포조망솔명현강저(t=6.14,P＜0.001).결론 대서엄중소상조기심기조직미소RNA-126표체하강,기가능삼여소상조기심기손해적조공병발휘보호작용.
Objective To observe the changes in the expressions of microRNA-126 in myocardial tissue and cardiac troponin Ⅰ (cTnⅠ) in serum of rats in the early stage of severe burn injury with analysis of their relationship, and to validate the relationship between microRNA-126 and myocardial damage in cellular level.Methods (1) Forty-eight SD rats were divided into sham injury group (n =8, without fluid therapy after sham injury) and burn injury group (n =40, inflicted with 30％ TBSA full-thickness scald on the back, hereinafter referred to as burn, and received intraperitoneally injection of lactic acid Ringer's solution) according to the random number table.Blood was collected from abdominal aorta of rats in sham injury group at post injury hour (PIH) 1, and then these 8 rats were sacrificed for obtaining left ventricular tissue.Blood was respectively collected from abdominal aorta of 8 rats in burn injury group at PIH 3, 6, 12, 24, and 48, and then they were sacrificed and the left ventricular tissue was obtained at each time point.The expression of microRNA-126 in myocardial tissue was assessed by real-time fluorescent quantitative RT-PCR.Serum level of cTnⅠ was assessed by ELISA.(2) Rat myocardial cell line H9C2 was divided into normal control group (NC, routinely cultured) , stimulation group (S) , negative transfection + stimulation group (NT + S), and transfection + stimulation group (T + S) according to the random number table.Cells in S group were treated with hypoxia for 24 h after being cultured with DMEM containing 10％ burn serum obtained from rats in burn injury group at PIH 6 in experiment (1).Cells in NT + S group and T + S group were respectively transfected with the negative control of microRNA mimics and microRNA-126 mimics for 24 h, and then were given the same treatment as that of S group.The expression of microRNA-126 in myocardial cells was determined by real-time fluorescent quantitative RT-PCR (with the sample number of 3).Cell counting kit 8 was used to examine the vitality of myocardial cell (with the sample number of 4, denoted as absorbance value).Apoptotic rate of myocardial cells was determined by flow cytometer (with the sample number of 3).Data were processed with one-way analysis of variance and LSD-t test.The relationship between microRNA-126 expression in myocardial tissue and serum level of cTnⅠ of rats was assessed by linear correlation analysis.Results (1) Compared with that of sham injury group at PIH 1, the expression levels of microRNA-126 in myocardial tissue of rats in burn injury group at PIH 3,6, 12, 24, and 48 were significantly decreased (with t values from 5.68 to 9.79, P values below 0.01) , reaching its nadir at PIH 24 (0.40 ±0.08).Compared with that of sham injury group at PIH 1 , the serum levels of cTnⅠ of rats in burn injury group at PIH 3, 6, 12, 24, and 48 were significantly increased (with t values from 6.68 to 12.79, P values below 0.01), peaking at PIH 12 [(1 035 ± 177) pg/mL].A significant negative correlation between the expression level of microRNA-126 in myocardial tissue and serum level of cTnⅠ was observed in rats of burn injury group at each time point (r =-0.797, P ＜0.001).(2) Compared with those of NC group, the microRNA-126 expression levels in myocardial cells of S group and T + S group were respectively decreased and increased (with t values respectively 4.57 and 5.73, P ＜ 0.05 or P ＜ 0.01) , the cell vitality levels were obviously decreased (with t values respectively 14.88 and 6.48, P values below 0.01) , and the apoptotic rates were significantly increased (with t values respectively 13.82 and 6.96, P values below 0.01).Compared with that in NT + S group, the microRNA-126 expression level in myocardial cells of T + S group was significantly increased (t =6.77, P ＜ 0.01) , the cell vitality level was obviously increased (t =8.23,P ＜0.001), and the apoptotic rate was significantly decreased (t =6.14, P ＜0.001).Conclusions Expression level of microRNA-126 in myocardial tissue of rat was decreased in the early stage of severe burn injury.It may participate in regulating myocardial damage and play a protective role.
