miR-29a影响eNOS蛋白表达参与血管内皮障碍机制研究

Mechanism of miR-29a affecting eNOS protein expression and participating in vascular endothelial dysfunction

目的 探究miR-29a抑制血管内皮细胞体外血管生成谷氨酰胺酶靶向细胞,初步寻求炎症因子诱发血管内皮功能障碍的相关机制。方法 选择人脐静脉内皮细胞作为研究细胞系,分别使用重组人肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、血清淀粉样蛋白A(serum amyloid A,SAA)对细胞系进行干预,采用实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,qRT-PCR)检测miRNA的表达变化,采用Western blot检测内皮型一氧化氮合酶(endothelial nitric oxide synthase,e NOS)表达变化,最后使用血管保护性药物丹参反向验证miR-29a表达及e NOS表达变化。结果 TNF-α与SAA的加入使miR-29a的表达水平较对照组显著提高(P<0.05)。qRT-PCR检测显示,随着加入炎症因子水平的升高,细胞系中miR-29a表达水平也呈现明显的升高趋势;以1.0ng/ml的TNF-α及SAA作为恒定剂量进行干预,观察炎症因子作用时间对miR-29a表达水平的影响,显示随着时间推移,miR-29a表达水平呈现升高趋势。采用Western blot检测TNF-α及SAA通过细胞系作用于eNOS蛋白表达情况,显示相较于对照组,加用TNF-α及SAA均可导致e NOS蛋白表达下调。随着丹参浓度的升高,miR-29a表达水平呈降低趋势,恒定浓度下miR-29a表达水平随时间呈现降低趋势。加入丹参后eNOS蛋白表达明显提升。结论 炎症因子TNF-α、SAA可通过诱导miR-29a表达参与血管内皮损伤进程,eNOS蛋白广泛参与该进程;应用血管保护性药物可在一定程度上抑制miR-29a的过表达,降低e NOS蛋白表达量,从而发挥保护血管的作用。

Objective To explore the inhibitory effect of miR-29a on in vitro angiogenesis of vascular endothelial cells by targeting cells with glutaminase,and preliminarily explore the relevant mechanisms of inflammatory factors inducing vascular endothelial dysfunction.Methods Human umbilical vein endothelial cells were selected as research cell lines,and the cell lines were intervened by recombinant human tumor necrosis factor-α(TNF-α)and serum amyloid A(SAA)respectively.The expression changes of miRNA were detected by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR).The expression changes of endothelial nitric oxide synthase(eNOS)were detected by Western blot.Finally,the vascular protective drug salvia miltiorrhiza was used to reverse verify the expression changes of miR-29a and eNOS.Results The addition of TNF-αand SAA significantly increased the expression level of miR-29a compared with control group(P<0.05).qRT-PCR showed that the higher the level of inflammatory factors,the higher the expression level of cell line miR-29a.With 1.0ng/ml TNF-αand SAA as a constant dose,it was found that the expression level of miR-29a increased gradually with time.Western blot showed that the addition of TNF-αand SAA would decrease the level of eNOS protein in the cell line.The expression level of miR-29a decreased with the increase of salvia miltiorrhiza concentration,and decreased with time at constant salvia miltiorrhiza concentration.After adding salvia miltiorrhiza,the expression of eNOS protein increased obviously.Conclusion Inflammatory factors TNF-αand SAA can participate in the process of vascular endothelial injury by inducing the expression of miR-29a,and eNOS protein is widely involved in this process.The application of vasoprotective drugs can inhibit the over-expression of miR-29a to some extent,reduce the expression of eNOS protein,and thus play a role in protecting blood vessels.