摘要目的 观察蛋白激酶B/哺乳动物雷帕霉素靶蛋白(Akt/mTOR)在高磷诱导的大鼠血管平滑肌细胞(VSMC)钙化模型中的表达及其对核心结合因子α1(Cbfα1)表达的调节.方法 体外培养的大鼠VSMC,分为正常磷组(1.3 mmol/L)和高磷组(2.6 mmol/L),培养7d后,观察细胞形态及茜素红染色细胞钙盐沉积,实时定量PCR检测Cbfα1、骨桥蛋白(OPN) mRNA水平,Western印迹检测磷酸化Akt(p-Akt)(ser473)、磷酸化mTOR(p-mTOR)(S2448)、Cbfα1和OPN蛋白的表达.检测到Akt、mTOR活化表达后,给予不同浓度的渥曼青霉素(5、10、30、50和100 nmol/L)和雷帕霉素(1、10和100 ng/ml)干预24、48 h后,检测Cbfα1、OPN、p-Akt和p-mTOR基因和蛋白表达,7d后观察VSMC钙盐沉积变化.结果 VSMC高磷干预7d后,与正常磷组相比,高磷组钙盐沉积明显,Cbfα1、OPN mRNA表达增高(均P＜0.05),p-Akt、p-mTOR、Cbfα1和OPN蛋白表达增高(均P＜0.05).渥曼青霉素、雷帕霉素干预24 h后,相对于高磷组,高磷+渥曼青霉素30、50、100 nmol/L组Cbfα1、OPNmRNA表达明显下调(均P＜0.05);高磷+雷帕霉素1、10和100 ng/ml组Cbfα1、OPN mRNA表达下调(均P＜0.05).渥曼青霉素、雷帕霉素干预48 h后,与高磷组比较,高磷+渥曼青霉素30、50、100 nmol/L)组p-Akt、Cbfα1和OPN蛋白表达明显下调(均P＜0.05);高磷+雷帕霉素1、10、100 ng/ml组,p-mTOR、Cbfα1和OPN蛋白表达呈剂量依赖性下调(均P＜0.05).VSMC培养7d后,与高磷组相比,高磷+渥曼青霉素和高磷+雷帕霉素组钙盐沉积明显减轻.结论 高磷可体外诱导大鼠VSMC钙化,Akt、mTOR参与了高磷诱导的大鼠VSMC钙化,并可能通过调控Cbfα1因子而起作用.

abstractsObjective To observe the expression of protein kinase B (Akt) / mammalian target of rapamycin (mTOR) induced by high phosphorus in rat vascular smooth muscle cells (VSMC) calcification model,and its modulation on the expression of core binding factor alpha 1 (Cbfα1).Methods Rat VSMC cells were cultured in vitro,and then divided into two groups:normal phosphorus group (Pi 1.3 mmol/L) and high phosphorus group (Pi 2.6 mmol/L).At day 7,calcium deposition was detected by Alizarin stain.The mRNA levels of Cbfα1 and osteopontin (OPN) were determined by real-time PCR.The protein expressions of p-Akt (ser473),p-mTOR (S2448),Cbfα1 and OPN were quantified by Western blot.Then,VSMC cultured with high phosphorus were treated with Akt inhibitor (Wortmannin) and mTOR inhibitor (Rapamycin) with different concentrations.After 24 h,the mRNA levels of Cbfα1 and OPN were determined and after 48 h,the protein expressions of p-Akt,p-mTOR,Cbfα1 and OPN were quantified.Also,at day 7,calcium deposition was also visualized by Alizarin stain.Results After 7 days,compared with normal phosphorus group,calcium deposition was more obvious in high phosphorus group.The mRNA expressions of Cbfα1 and OPN increased significantly and the protein expressions of p-Akt,p-mTOR,Cbfα1 and OPN up-regulated significantly in high phosphorus group (all P ＜ 0.05).After treated with Wortmannin or Rapamycin for 24 h,compared with high phosphorus group,the mRNA expressions of Cbfα1 and OPN decreased significantly in high phosphorus + Wortmannin (30,50 and 100 nmol/L) groups (all P ＜ 0.05) and high phosphorus + Rapamycin (1,10 and 100 ng/ml) groups (all P ＜ 0.05).After treated with Wortmannin or Rapamycin for 48 h,compared with high phosphorus group,the protein expressions of p-Akt,Cbfα1 and OPN down-regulated significantly in high phosphorus + Wortmannin (30,50 and 100 nmol/L) groups (all P ＜ 0.05).It showed a dose-dependent down-regulation of p-mTOR,Cbfoα1 and OPN in high phosphorus + Rapamycin (1,10,100 ng/ml) groups (all P ＜0.05).After 7 days,compared with high phosphorus group,calcium deposition decresased significantly in high phosphorus + Wortmannin and high phosphorus + Rapamycin groups.Conclusions High phosphorus can induce VSMC calcification.Akt and mTOR are involved in VSMC calcification induced by high phosphorus through the activation of Cbfα1.