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目的 探讨微小RNA-1(miR-1)在高糖培养所致大鼠心肌纤维化中的作用途径.方法 取1~3日龄SD大鼠心尖组织培养原代心肌成纤维细胞,传代至3~4代细胞后被随机分为正常糖空病毒组(CON+ Lv-Vehicle组)、正常糖miR-1沉默组(CON+Lv-miR1组)、高糖空病毒组(HG+Lv-Vehicle组)、高糖miR-1沉默组(HG+Lv-miR1组)、高糖空病毒抑制剂组(HG+Lv-Vehicle+CC组)、高糖miR-1沉默抑制剂组(HG+Lv-miR1+CC组).将细胞分别置于葡萄糖5.5 mmol/L(正常糖)和25.0 mmol/L(高糖)的DMEM培养基中,接种含miR-1沉默序列的慢病毒载体或慢病毒;抑制剂组于取样前12 h加入20 μmol/L腺苷酸活化蛋白激酶(AMPK)抑制剂Compound C.采用蛋白质免疫印迹试验(Western Blot)检测磷酸化AMPK(p-AMPK)、胶原蛋白Ⅰ和Ⅲ、基质金属蛋白酶(MMP-2、MMP-9)、自噬流相关蛋白〔微管相关蛋白1轻链3B-Ⅱ(LC3B-Ⅱ)、死骨片重组蛋白1(p62/SQSTM1)〕的蛋白表达.结果 体外培养大鼠心肌成纤维细胞的纯度达97%.与CON+Lv-Vehicle组比较,CON+Lv-miR1组p-AMPK表达无明显变化,HG+Lv-Vehicle组p-AMPK表达明显降低(p-AMPK/t-AMPK:44.72±3.29比100.00±7.77,1<0.01);HG+Lv-miR1组p-AMPK表达较HG+Lv-Vehicle组明显增高(p-AMPK/t-AMPK:60.52±5.16比44.72±3.29,1<0.05).与HG+Lv-Vehicle组比较,HG+Lv-miR1组胶原蛋白、MMP、LC3B-Ⅱ和p62/SQSTM1表达均明显降低;给予AMPK抑制剂后胶原蛋白、MMP、LC3B-Ⅱ、p62/SQSTM1表达均明显增高(HG+Lv-Vehicle+CC组与HG+Lv-Vehicle组比较:胶原蛋白Ⅰ/β-actin为158.74±13.21比100.00±7.64,胶原蛋白Ⅲ/β-actin为177.38±17.31比100.00±5.18,MMP-2/β-actin为130.09±14.31比100.00±10.47,MMP-9/β-actin 为215.54±20.92比100.00±11.28,LC3B-Ⅱ/β-actin 为159.34±13.83比100.00±6.44,p62/SQSTM1/β-actin为201.01±24.02比100.00±8.62;HG+Lv-miR1+CC组与HG+Lv-miR1组比较:胶原蛋白Ⅰ/β-actin为108.69±9.93比80.83±7.24,胶原蛋白Ⅲ/β-actin为127.68± 10.46比81.56±9.97,MMP-2/β-actin为106.66±10.21比74.80±7.43,MMP-9/β-actin为145.65±11.56比74.63±10.55,LC3B-Ⅱ/β-actin 为150.15±13.28比22.98±2.87,p62/SQSTM1/β-actin 为130.48±10.74比49.90±2.27,均1<0.05).结论 miR-1基因沉默对高糖培养致大鼠心肌成纤维细胞纤维化具有抑制作用,其机制可能与上调AMPK磷酸化表达、恢复成纤维细胞自噬流有关.

Objective To investigate the role of microRNA-1 (miR-1) in cardiac fibroblasts induced by high glucose in rats. Methods The primary fibroblasts were cultured from the apical tissue of 1-3 day-old Sprague-Dawley (SD) rats. The cells which were passaged to generation 3 or 4, were randomly divided into normal glucose+lentivector-vehicle group (CON+Lv-Vehicle group), normal glucose+lentivector-miR-1 group (CON+Lv-miR1 group), high glucose+lentivector-vehicle group (HG+Lv-Vehicle group), high glucose+lentivector-miR-1 group (HG+Lv-miR1 group), high glucose+Lv-Vehicle+inhibitor group (HG+Lv-Vehicle+CC group), and high glucose+lentivector-miR-1+inhibitor group (HG+Lv-miR1+CC group). The myocardial fibroblasts were cultured in the concentration of 5.5 mmol/L glucose (normal glucose) or 25.0 mmol/L (high glucose) DMEM medium. Then lentiviral vector containing miR-1 silent sequence or the same volume of lentiviral vector was inoculated into the cells. The AMP activated protein kinase (AMPK) inhibitor Compound C (20 μmol/L) was added to the medium at 12 hours before sampling in inhibitor groups. The expression of phosphorylation of AMPK (p-AMPK), collagenⅠandⅢ, matrix metalloproteinase (MMP-2, MMP-9), and autophagy flux related protein LC3B-Ⅱ and p62/SQSTM1 were measured by Western Blot. Results The purity of rat myocardial fibroblasts in vitro was 97%. Compared with CON+Lv-Vehicle group, there was no significant difference in the expression of p-AMPK in CON+Lv-miR1 group, the expression of p-AMPK in HG+Lv-Vehicle group was significantly decreased (p-AMPK/t-AMPK: 44.72±3.29 vs. 100.00±7.77, 1 < 0.01). The expression of p-AMPK in HG+Lv-miR1 group was higher than that in HG+Lv-Vehicle group (p-AMPK/t-AMPK:60.52±5.16 vs. 44.72±3.29, 1 < 0.05). Compared with HG+Lv-Vehicle group, the expressions of collagen, MMP, LC3B-Ⅱand p62/SQSTM1 in HG+Lv-miR1 group were significantly decreased; after the treatment with AMPK inhibitor, the expressions of collagen, MMP, LC3B-Ⅱ, p62/SQSTM1 were significantly increased (HG+Lv-Vehicle+CC group vs. HG+Lv-Vehicle group: collagen Ⅰ/β-actin: 158.74±13.21 vs. 100.00±7.64, collagenⅢ/β-actin: 177.38± 17.31 vs. 100.00±5.18, MMP-2/β-actin: 130.09±14.31 vs. 100.00±10.47, MMP-9/β-actin: 215.54±20.92 vs. 100.00±11.28, LC3B-Ⅱ/β-actin: 159.34±13.83 vs. 100.00±6.44, p62/SQSTM1/β-actin: 201.01±24.02 vs. 100.00±8.62; HG+Lv-miR1+CC group vs. HG+Lv-miR1 group: collagenⅠ/β-actin: 108.69±9.93 vs. 80.83±7.24, collagenⅢ/β-actin: 127.68±10.46 vs. 81.56±9.97, MMP-2/β-actin: 106.66±10.21 vs. 74.80±7.43, MMP-9/β-actin: 145.65±11.56 vs. 74.63±10.55, LC3B-Ⅱ/β-actin: 150.15±13.28 vs. 22.98±2.87, p62/SQSTM1/β-actin: 130.48±10.74 vs. 49.90±2.27, all 1 < 0.05). Conclusion miR-1 gene silencing inhibits myocardial fibrosis induced by high glucose, its mechanism may be related to the up-regulation of p-AMPK, which can recover autophagy flux.