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微小RNA-1在高糖培养下心肌成纤维细胞致纤维化中的调控作用 被引量:3

Role of microRNA-1 on fibrosis induced by high glucose cultured cardiac fibroblasts
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摘要 目的探讨微小RNA-1(miR-1)在高糖培养下心肌成纤维细胞致纤维化中的作用。方法取1-3日龄SD大鼠心尖组织培养原代成纤维细胞,选用3-4代细胞并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)4组,分别置于含葡萄糖5.5mmol/L(正常糖)或25mmol/L(高糖)的DMEM培养基中,并分别接种含miR-1沉默序列的慢病毒或慢病毒载体,于12h后更换新鲜培养基。接种病毒3d后病毒转染效率达90%时,采用实时定量反转录-聚合酶链反应(qRT-PCR)检测miR-1表达,酶联免疫吸附试验(ELISA)检测胶原蛋白Ⅰ和Ⅲ的分泌量,蛋白质免疫印迹试验(Western Blot)检测胶原蛋白Ⅰ和Ⅲ、基质金属蛋白酶(MMP-2、MMP-9)及自噬相关蛋白微管相关蛋白l轻链3B-Ⅱ(LC3B-Ⅱ)、死骨片重组蛋白1(P62/SQSTM1)、组织蛋白酶D(Cathepsin D)的蛋白表达。结果与CON+Lv-Vehicle组比较,CON+Lv-miR1组各指标差异均无统计学意义;HG+Lv-Vehicle组miR-1表达明显升高(2^△△Ct:1.82±0.17比1.00±0.04),胶原蛋白Ⅰ和Ⅲ的分泌量(mg/L:14.55±0.33比7.28±0.22,157.50±13.22比61.25±8.54)及表达量(灰度值:432.35±56.00比100.00±15.00,320.35±47.00比100.00±15.00)明显升高,MMP-2、MMP-9、LC3B-Ⅱ、P62/SQSTM1蛋白表达明显增高(灰度值:249.0±21.0比100.0±15.0,142.3±20.0比100.0±16.0,178±19比100±14,378.3±20.0比100.0±15.0),而CathepsinD表达明显降低(灰度值:60±14比100±10),差异均有统计学意义(均P〈0.01)。与HG+Lv-Vehicle组比较,HG+Lv-miR1组miR-1表达明显降低(2^△△Ct:1.21±0.10比1.82±0.17),胶原蛋白Ⅰ和Ⅲ的分泌量(mg/L:10.68±0.54比14.55±0.33,87.25±13.55比157� Objective To investigate the effect of microRNA-1 (miR-1) on the fibrosis of cardiac fibroblasts induced by high glucose. Methods The primary cultured fibroblasts from 1-3 days old Sprague-Dawley (SD) rats, were randomly divided into 4 groups (n = 3): normal glucose + lentivector-vehicle (CON+Lv-Vehiele group), normal glucose + lentivector-miR-1 (CON+Lv-miR1 group), high glucose + lentivector-vehicle (HG+Lv-Vehicle group), high glucose + lentivector-miR-1 (HG+Lv-miR1 group). Fibroblasts were cultured in glucose concentration 5.5 mmol/L and 25 mmol/L DMEM culture, and were injected lentiviral vector carrying miR-1 silencer sequence or the same volume of lentiviral vector. After 12 hours, the medium was replaced with fresh complete medium. After 3 days when transfection efficiency was up to 90%, the cellular miR-1 content was detected by real-time reverse transcription-polymerase chainreaction (qRT-PCR). The secretion of collagen Ⅰ and Ⅲ were measured by enzyme linked immunosorbent assay (ELISA). Expression of collagen Ⅰ and Ⅲ, matrix metalloproteinase 2 and 9 (MMP-2, MMP-9), autophagy related protein LC3B-Ⅱ, P62/SQSTM1 and Cathepsin D were assessed by Western Blot. Results Compared with the CON+Lv- Vehicle group, the content of miRNA in the CON+Lv-miR1 group had no statistical significance. Compared with the CON+Lv-Vehicle group, high glucose increased the amount of miR-1 (2^△△Ct: 1.82±0.17 vs. 1.00±0.04), collagen Ⅰ and Ⅲ secretion (mg/L: 14.55 ±0.33 vs. 7.28 ± 0.22, 157.50 ±13.22 vs. 61.25 ± 8.54) and expression (gray value: 432.35 ± 56.00 vs. 100.00 ± 15.00, 320.35 ± 47.00 vs. 1130.00 ± 15.00), the level of MMP-2, MMP-9 and the expression of autophagy related protein LC3B-Ⅱ and P62/SQSTM1 (gray value: 249.0±21.0 vs. 100.0±15.0, 142.3±20.0 vs. 100.0 ± 16.0, 178 ± 19 vs. 100 ± 14, 378.3 ± 20.0 vs. 100.0 ± 15.0), decreased the expression of lysosomal associated protein Cathepsin D (gray v
作者 王安 孙波 仇佳 乔世刚 王琛 Wang An, Sun Bo, Qiu Jia, Qiao Shigang, Wang Chen (Department of Anesthesiology, Second Affiliated Hospital, Soochow University, Suzhou 215004, Jiangsu, China (Wang A, Sun B, Qiu J, Qiao SG, Wang C); Department of Anesthesiology, Suzhou Science and Technology Town Hospital, Suzhou 215153, Jiangsu, China (Qiao SG, Wang C); Department of Anesthesiology, Suzhou Hospital, Nanjing Medical University, Suzhou 215001, Jiangsu, China (Qiao SG, Wang C))
出处 《中华危重病急救医学》 CAS CSCD 北大核心 2016年第12期1113-1117,共5页 Chinese Critical Care Medicine
基金 江苏省自然科学基金面上项目(BK20141187) 江苏省苏州市科技计划项目(SS201613,SYS201473) 苏州大学科研预研基金项目(SDY2015A18)
关键词 高糖损伤 心肌成纤维细胞 纤维化 微小RNA 自噬 High glucose damage Cardiac fibroblast Fibrosis MicroRNA Autophagy
作者简介 通讯作者:王琛,Email:wangchen1791@163.com
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