Objective To investigate the mechanism of action of advanced glycation end products(AGEs) in regulating the receptor of AGEs(RAGE) for promoting calcification of human femoral arterial smooth muscle cells.Methods This study was carried out between January 2014 and March 2015.The tunica media of femoral artery was cut into tissue blocks of 1-2 mm in length for adherent culture.We selected the cultured smooth muscle cells of 3-8 generation for the experiment and divided them into 4 groups for further culture,group A with DMEM culture medium;group B using DMEM+10 mmol/L β-sodium phosphate;group C using DMEM+10 mmol/L β-sodium phosphate+AGEs 20 mg/L;and group D with DMEM+10 mmol/L β-sodium phosphate+AGEs 40 mg/L.Cells in each group were intervened for 96 hours.Three pairs of random RAGE siRNA were synthesized,and used to transfect the smooth muscle cells of group C with a concentration of 50 nmol/L.The efficacy of siRNA was measured 24 h later.The sequences with high transfection efficiency were selected for experiment.The calcium deposition in smooth muscle cells were observed by Von Kossa staining before and after RAGE-specific siRNA transfection.The expression levels of RAGE,β-catenin,and osteoprotegerin(OPG) were detected by Western blotting.Results In the non-transfected cells,there was no calcified plaque in group A,while group B showed obvious calcification plaques;in groups C and D,the number of cytoplasm and nucleus of the smooth muscle cells in red increased and calcified plaques increased significantly.After RAGE-specific siRNA transfection,the number of cytoplasm and nucleus of the smooth muscle cells in red in group C was decreased compared with that before transfection,and the formation of calcified plaque was inhibited.Before RAGE-specific siRNA transfection,the expression levels of RAGE,β-catenin,and OPG in smooth muscle cells differed significantly among the groups(P<0.05),specifically,the expression levels of RAGE,β-catenin,and OPG in smooth muscle cells were higher in group C than in group B,and they were higher in group D than in groups B and C(P<0.05).The expressions of RAGE,β-catenin,and OPG in smooth muscle cells in group C were lower after RAGE-specific siRNA transfection compared with those before transfection(P<0.01).Conclusion AGEs mediate smooth muscle cells calcification by regulating RAGE,the mechanism may be that RAGE activates the Wnt/ β-catenin signaling pathway and by which the expression level of β-catenin downstream protein OPG is increased.%目的 探讨晚期糖基化终末产物(AGEs)调控其受体促进人股动脉平滑肌细胞钙化的机制.方法 2014年1月-2015年3月,将股动脉中膜平滑肌层剪成1~2 mm组织块培养,选取3~8代平滑肌细胞用于实验.将平滑肌细胞分为4组,A组单纯于DMEM培养基中培养,B组于含10 mmol/L β磷酸甘油钠的DMEM培养基中培养,C、D组分别于含10 mmol/L β磷酸甘油钠的DMEM培养基中加入20、40 mg/L AGEs,各组平滑肌细胞干预培养96 h.合成3对随机AGEs受体(RAGE)siRNA,以50 nmol/L转染C组平滑肌细胞,24 h后进行siRNA沉默效果检测,选取转染效率较高的序列用于实验.分别取转染前后平滑肌细胞,采用Von Kossa染色实验观察平滑肌细胞内钙沉积情况,采用Western blotting法检测RAGE、β-catenin、骨保护素(OPG)表达水平.结果 未转染的平滑肌细胞中,A组无钙化斑块形成,B组可见明显的钙化斑块;C、D组细胞质、细胞核呈现粉红色的数量增加,钙化斑块明显增多.经转染后,C组平滑肌细胞细胞质、细胞核呈现粉红色的数量较未转染细胞减少,钙化斑块形成受到抑制.未转染的各组平滑肌细胞RAGE、β-catenin、OPG表达水平比较,差异均有统计学意义(P<0.05);其中,C组平滑肌细胞RAGE、β-catenin、OPG表达水平高于B组,D组平滑肌细胞RAGE、β-catenin、OPG表达水平高于B、C组(P<0.05).C组平滑肌细胞转染后RAGE、β-catenin、OPG表达水平均低于转染前(P<0.01).结论 AGEs通过与其受体结合而介导平滑肌细胞钙化,其机制可能为RAGE激活Wnt/β-catenin信号通路,使β-catenin下游蛋白OPG表达水平升高.
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