【摘要】 目的:探讨淫羊藿-仙茅(二仙)药对在miRNA水平保护高糖介导成骨细胞的作用机制。方法:建立高糖介导的成骨细胞模型;MTT法检测成骨细胞活性;生物信息学算法预测miR-144靶基因。RT-PCR检测miR-144和靶基因的mRNA表达。构建荧光素酶报告质粒,分析荧光素酶活性。将miR-144模拟物和抑制剂通过Lipofectamine 2000转染进高糖介导的成骨细胞。免疫印迹法检测胰岛素受体底物1(IRS1)蛋白表达。结果:二仙药对能提高高糖介导的成骨细胞活性,促进增殖;生物信息学提示IRS1是miR-144的靶基因。与对照组比较,模型组miR-144表达显著升高(P<0.05),IRS1 mRNA表达水平显著降低(P<0.01);二仙药对组抑制了这种变化。与阴性对照组比较,miR-144模拟物可显著抑制与IRS1 3’-UTR融合的荧光素酶报告基因的活性。模型组IRS1mRNA表达下调,二仙药对组表达相反,说明二仙药对能够上调转染miR-144模拟物或抑制剂的高糖成骨细胞中的IRS1 mRNA表达。在高糖成骨细胞模型中,转染miR-144模拟物组的IRS1蛋白表达均显著增加(P<0.01),转染miR-144抑制剂后该蛋白表达显著降低(P<0.01)。二仙药对治疗后,IRS1蛋白表达显著增加。结论:miR-144在高糖介导的成骨细胞中能调控IRS1表达。二仙药对能下调或维持miR-144水平,增加IRS1 mRNA和蛋白表达。更多还原

【Abstract】 Objective: To explore the mechanism of action of herba epimedii-curculigo orchioides(Erxian) couplet medicines on the protection of high glucose-medicated osteoblasts at the miRNA level. Methods: The osteoblast model medicated by hyperglycemia was established. MTT assay was used to detect osteoblast activity, the bioinformatics algorithm was used to predict the miR-144 target gene. RT-PCR was used to detect mRNA expression of miR-144 and target genes. The luciferase reporter plasmid was constructed to analyze luciferase activity. The miR-144 mimics and inhibitors were transfected into high glucose-medicated osteoblasts via lipofectamine 2000. IRS1 protein expression was detected by immunoblotting. Results:Erxian couplet medicines could increase the activity of osteoblasts mediated by hyperglycemia and promote proliferation.Bioinformatics suggested that IRS1 was the target gene of miR-144. Compared with the control group, the expression of miR-144 was significantly increased in the model group(P<0.05), and the expression level of IRS1 mRNA was significantly decreased(P<0.01); and the Erxian couplet medicines group inhibited this change. The miR-144 mimics significantly inhibited the activity of the luciferase reporter gene fused with IRS1 3’-UTR compared to the negative control. The expression of IRS1 mRNA was down regulated in the model group, while the expression showed opposite change in Erxian couplet medicines group. Erxian couplet medicines could up regulate IRS1 mRNA expression in high glucose-medicated osteoblast transfected with miR-144 mimics or inhibitors. In the high glucose-medicated osteoblast model, the protein expression of IRS1 was increased significantly in the miR-144 mimics group(P<0.01), and the protein expression was significantly decreased after transfection with miR-144 inhibitor(P<0.01). The protein expression was significantly increased after treatment with Erxian couplet medicines(P<0.01). Conclusion:miR-144 can regulate the expression of IRS1 in high glucose-mediated osteoblasts. Erxian couplet medicines could down-regulate or maintain the miR-144 levels and increase IRS1 mRNA and protein expressions.更多还原