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目的:探讨微小RNA-1（miR-1）对缺氧/复氧（H/R）心肌细胞凋亡的调节作用。方法:体外培养大鼠胚胎心脏组织来源心肌细胞株H9c2，将处于对数生长期的细胞分为空白对照组、H/R组、miR-1模拟物（mimics）+H/R组、miR-1抑制剂反义寡核苷酸（ASO）+H/R组和微小RNA阴性对照片段（miRNA NC）+H/R组。用含低浓度胎牛血清（FBS）的低糖DMEM培养基作为缺氧条件下的培养基，置于37 ℃密闭无氧培养箱中（95% Nn 2和5% COn 2）培养12 h后，再换取新鲜的含5% FBS的高糖DMEM培养基，置于37 ℃密闭培养箱中培养，建立心肌细胞H/R模型；空白对照组用含10% FBS的高糖DMEM培养基，于37 ℃、5% COn 2培养箱中培养。miR-1 mimics+H/R组、miR-1 ASO+H/R组、miRNA NC+H/R组分别于制模前在高糖培养基中加入相应转染物，终浓度均为50 nmol/L；空白对照组和H/R组不予转染处理。制模完成后，采用实时荧光定量聚合酶链反应（qPCR）检测细胞miR-1的表达水平；采用蛋白质免疫印迹试验（Western blotting）检测细胞凋亡相关蛋白天冬氨酸特异性半胱氨酸蛋白酶9（caspase-9）、Bcl-2和Bax的表达水平；应用流式细胞仪检测心肌细胞凋亡情况。n 结果:与空白对照组相比，H/R组心肌细胞中miR-1表达水平、caspase-9和Bax的蛋白表达水平、细胞凋亡率均明显升高，而Bcl-2表达水平明显下降，说明H/R心肌细胞中miR-1表达增加，凋亡水平升高。与H/R组相比，miR-1 mimics+H/R组心肌细胞中miR-1表达水平、caspase-9和Bax蛋白表达水平、细胞凋亡率均进一步升高〔miR-1（2n -ΔΔCt）：11.59±1.48比2.57±0.38，caspase-9蛋白（caspase-9/β-actin）：2.59±0.12比1.56±0.20，Bax蛋白（Bax/β-actin）：4.09±0.38比1.97±0.13，细胞凋亡率：（25.23±0.87）%比（17.86±0.73）%，均n P<0.01〕，而Bcl-2蛋白表达水平则进一步下降（Bcl-2/β-actin：0.37±0.02比0.49±0.03，n P<0.01）；miR-1 ASO+H/R组miR-1表达水平、caspase-9和Bax蛋白表达水平、细胞凋亡率显著下降〔miR-1（2n -ΔΔCt）：1.16±0.06比2.57±0.38，caspase-9蛋白（caspase-9/β-actin）：1.05±0.24比1.56±0.20，Bax蛋白（Bax/β-actin）：0.93±0.11比1.97±0.13，细胞凋亡率：（11.19±0.85）%比（17.86±0.73）%，均n P<0.05〕，而Bcl-2的表达水平则显著升高（Bcl-2/β-actin：0.84±0.17比0.49±0.03，n P<0.05）；miRNA NC+H/R组miR-1表达，caspase-9、Bax和Bcl-2蛋白表达以及细胞凋亡率与H/R组比较差异均无统计学意义。n 结论:H/R心肌细胞中miR-1表达增加、凋亡水平升高，且miR-1可加剧心肌细胞凋亡。“,”Objective:To investigate the changes of cardiomyocyte apoptosis after hypoxia/reoxygenation (H/R) regulated by microRNA-1 (miR-1).Methods:Cardiomyocyte strain H9c2 derived from rat embryonic heart tissue were cultured n in vitro. The cells in logarithmic growth phase were divided into blank control group, H/R group, miR-1 mimics+H/R group, miR-1 inhibitor antisense oligonucleotide (ASO)+H/R group and microRNA negative control fragment (miRNA NC)+H/R group. The low sugar DMEM medium containing low concentration of fetal bovine serum (FBS) was used as the medium under anoxic condition. After being cultured in a closed anaerobic incubator at 37 ℃ (95% Nn 2 and 5% COn 2) for 12 hours, the cells were cultured with the fresh high sugar DMEM medium containing 5% FBS in a closed incubator at 37 ℃ for reproducing cardiomyocyte H/R model. The blank control group was cultured in high glucose DMEM medium containing 10% FBS in 37 ℃ and 5% COn 2 incubator. In miR-1 mimics+H/R group, miR-1 ASO+H/R group and miRNA NC+H/R group, the corresponding transfectants were mixed in high glucose DMEM medium and transfected into cells before H/R model was established, and the final concentration was 50 nmol/L. The blank control group and H/R group were added with DMEM medium at the same time. After the establishment of the model, the expression level of miR-1 was detected by real-time fluorescence quantitative polymerase chain reaction (qPCR). The expression levels of apoptosis-related proteins caspase-9, Bcl-2 and Bax were detected by Western blotting, and cardiomyocyte apoptosis was detected by flow cytometry.n Results:Compared with the blank control group, the expression levels of miR-1, caspase-9 and Bax protein and the apoptosis rate of cardiomyocytes were significantly increased, while the expression level of Bcl-2 was significantly decreased, which indicated that the expression of miR-1 and the level of apoptosis were increased in H/R group. Compared with H/R group, the expressions of miR-1, caspase-9 and Bax and the apoptosis rate of cardiomyocytes in miR-1 mimics+H/R group were further increased [miR-1 (2n -ΔΔCt): 11.59±1.48 vs. 2.57±0.38, caspase-9 protein (caspase-9/β-actin): 2.59±0.12 vs. 1.56±0.20, Bax protein (Bax/β-actin): 4.09±0.38 vs. 1.97±0.13, apoptosis rate: (25.23±0.87)% vs. (17.86±0.73)%, alln P < 0.01], while the expression of Bcl-2 was decreased (Bcl-2/β-actin: 0.37±0.02 vs. 0.49±0.03, n P < 0.01). The expressions of miR-1, caspase-9 and Bax and the apoptosis rate were significantly decreased in miR-1 ASO+H/R group [miR-1 (2 n -ΔΔCt): 1.16±0.06 vs. 2.57±0.38, caspase-9 protein (caspase-9/β-actin): 1.05±0.24 vs. 1.56±0.20, Bax protein (Bax/β-actin): 0.93±0.11 vs. 1.97±0.13, apoptosis rate: (11.19±0.85)% vs. (17.86±0.73)%, alln P < 0.05], while the expression of Bcl-2 was increased (Bcl-2/β-actin: 0.84±0.17 vs. 0.49±0.03, n P < 0.05). There was no significant difference in miR-1 expression, caspase-9, Bax and Bcl-2 protein expressions, and apoptosis rate between H/R+miRNA NC group and H/R group.n Conclusion:The expression of miR-1 and level of apoptosis were increased in H/R cells, and miR-1 could aggravate cardiomyocyte apoptosis.