【摘要】 目的探讨微小RNA-21(miR-21)对缺氧/复氧(H/R)损伤后大鼠肾小管上皮细胞(NRK-52E细胞)的影响。方法体外培养NRK-52E细胞,将其随机分为4组。miR-21激活剂(pre-miR-21)+H/R组、miR-21拮抗剂(antagomiR-21)+H/R组、模型组建立H/R损伤模型,正常组常规培养。pre-miR-21+H/R组、antagomiR-21+H/R组在缺氧处理前24 h分别加入终浓度均为30 nmol/L的pre-miR-21、antagomiR-21预处理。用CCK-8法检测细胞活力,用流式细胞术检测细胞凋亡能力,用RT-PCR检测细胞内miR-21、程序性细胞凋亡因子4(PDCD4)mRNA表达,用Western blotting法检测细胞内半胱氨酸天冬氨酸蛋白酶3(Caspase-3)蛋白表达,用化学比色法、终点比色法检测细胞抗氧化酶活性和总抗氧化能力,用ELISA法检测细胞培养上清液中炎症细胞因子。结果与正常组比较,模型组细胞活力低、凋亡率高、miR-21相对表达量高、PDCD4 mRNA相对表达量低、Caspase-3蛋白剪切片段P17相对表达量高、抗氧化酶活性和总抗氧化能力低、炎症细胞因子水平高(P均<0.05);与模型组比较,pre-miR-21+H/R组以上指标均改善(P均<0.05)。antagomiR-21+H/R组除miR-21相对表达量外其他指标与模型组比较差异无统计学意义(P均>0.05)。结论 miR-21可抑制H/R损伤后大鼠NRK-52E细胞凋亡,增加其抗氧化能力,并减轻炎症反应。更多还原

【Abstract】 Objective To investigate the effect of microRNA-21(miR-21) on renal tubular epithelial cells(NRK-52E) injured by hypoxia/reoxygenation(H/R) in rats.Methods NRK-52 E cells were cultured in vitro and randomly divided into 4 groups:pre-miR-21+H/R group,antagomiR-21+H/R group,model group,and normal group.The H/R injury models were established in the model group,and the cells in the normal group were routinely cultured.The cells in the pre-miR-21+H/R group and the antagomiR-21+H/R group were pre-treated with pre-miR-21 and antagomiR-21 at a final concentration of 30 nmol/L 24 h before hypoxia,respectively.Cell viability was detected by CCK-8,the apoptosis rate was measured by flow cytometry,miR-21 and PDCD4 mRNA expression levels were detected by RT-PCR,and Caspase-3 protein was detected by Western blotting.The activity of antioxidant enzymes and total antioxidant capacity of cells were detected by chemical colorimetry and endpoint colorimetry,and inflammatory cytokines in supernatant of cell culture were detected by ELISA.Results Compared with the normal group,the model group had lower cell viability,higher apoptosis rate,higher relative expression level of miR-21,lower relative expression level of PDCD4 mRNA,higher relative expression level of Caspase-3 protein P17,lower antioxidant enzyme activity and total antioxidant capacity,and higher level of inflammatory cytokines(all P<0.05).Compared with the model group,the pre-miR-21+H/R group had higher cell viability,lower apoptosis rate,lower relative expression level of miR-21,higher relative expression level of PDCD4 mRNA,lower relative expression level of Caspase-3 protein P17,higher antioxidant enzyme activity and total antioxidant capacity,and lower level of inflammatory cytokines(all P<0.05).There was no significant difference between antagomiR-21+H/R group and model group in the indicators except the relative expression of miR-21(all P>0.05).Conclusion MiR-21 can inhibit the apoptosis of NRK-52 E cells damaged by H/R,increase the antioxidant capacity of NRK-52 E cells,and alleviate inflammation.更多还原