抵抗素诱导血管平滑肌细胞表型转化的作用及机制研究

Effects of resistin on phenotypic transition of vascular smooth muscle cells and the underlying mechanism

目的研究抵抗素对血管平滑肌细胞表型转化的影响,探索其可能的分子机制。方法取生长状态良好的人血管平滑肌细胞(HVSMCs),使用不含胎牛血清的高糖细胞培养基培养24h进行同步化,各组分别用抵抗素(50μg/L)和二甲双胍(2mmol/L)处理细胞,然后通过流式细胞术检测细胞周期变化,实时荧光定量聚合酶链反应(RT-PCR)法检测α-平滑肌肌动蛋白(α-SMA)及骨桥蛋白(OPN)的基因表达,蛋白质免疫印迹(Westernblot)检测信号通路蛋白腺苷酸活化蛋白激酶(AMPK)、哺乳动物雷帕霉素靶蛋白(mTOR)及其磷酸化(p-AMPK、p-mTOR)表达水平,细胞周期蛋白D1(CyclinD1)表达水平。结果与对照组比较,抵抗素(Res)组G0/G1期细胞比例降低,S期细胞比例增加;α-SMA基因表达降低,OPN基因表达升高;p-AMPK表达降低,p-mTOR及CyclinD1表达增加(P<0.05)。与抵抗素组比较,经二甲双胍预处理后(Res+Met组)可以逆转以上变化。结论抵抗素可能通过抑制AMPK/mTOR信号通路,上调细胞周期蛋白CyclinD1表达,促进细胞进入S期,诱导血管平滑肌细胞表型转化。二甲双胍可减轻抵抗素对血管平滑肌细胞表型转换的影响,有潜在抑制冠脉移植血管增殖、重塑及减轻狭窄的作用。

Objective To study the effect of resistin on the phenotypic transition of vascular smooth muscle cells and explore its underlying molecular mechanism.Methods Human vascular smooth muscle cells(HVSMCs)in good growth state were cultured with high glucose medium without fetal bovine serum(DMEM)for 24 h to synchronize cell cycles.Cells in each group were treated with resistin(50μg/L)and metformin(2 mmol/L),respectively,and then the cell cycle changes were assessed by flow cytometry.Real-time fluorescence quantitative PCR(RT-PCR)was used to detect the expression ofα-smooth muscle actin(α-SMA)and os⁃teopontin(OPN).Western blot was used to detect the expression levels of signaling pathway proteins AMPK,mTOR,p-AMPK,pmTOR,and Cyclin D1.Results Compared with the control group,the proportion of cells in G0/G1 phase decreased and the propor⁃tion of cells in S phase increased in the resistin(Res)group.α-SMA gene expression decreased but OPN gene expression increased.The expression of p-AMPK decreased,while the expression of p-mTOR and Cyclin D1 increased(P<0.05).Compared with resistin group,metformin pretreatment(Res+Met group)could reverse the above changes.Conclusion Resistin may up-regulate the expres⁃sion of Cyclin D1 by inhibiting AMPK/mTOR signaling pathway,promote cells to enter S phase,and thus induce phenotypic transfor⁃mation of vascular smooth muscle cells.Metformin can reduce the effect of resistin on the phenotype conversion of vascular smooth mus⁃cle cells,and thus has the potential inhibit effect on the proliferation,remodeling and stenosis of coronary artery grafts.