Expression of microRNA-126 in myocardial tissue of rats in the early stage of severe burn injury and its relation with myocardial damage
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摘要: 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1 h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6 h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1 h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1 h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。Abstract: 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 (witht 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 (witht 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 (witht values respectively 4.57 and 5.73,P <0.05 orP <0.01), the cell vitality levels were obviously decreased (witht values respectively 14.88 and 6.48,P values below 0.01), and the apoptotic rates were significantly increased (witht 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.-
Key words:
- Burns /
- Anoxia /
- Apoptosis /
- Troponin I /
- MicroRNA-126 /
- Cell vitality
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