钙磷诱导大鼠血管平滑肌细胞钙化的机制研究

Investigation for the Mechanism of Vascular Smooth Muscle Cell Calcification Induced by Calcium and Phosphorus in Experimental Rats

目的:探讨氧化应激损伤在钙磷诱导的血管平滑肌细胞(VSMCs)钙化过程中的作用机制。方法:采用氯化钙(Ca Cl2)联合β-甘油磷酸钠(β-GP)建立大鼠VSMCs钙化模型。实验分为4组,对照组、钙化组、钙化+过氧化氢(H2O2)组和钙化+过氧化氢酶组。8天后分别通过茜素红S染色法和邻甲酚肽络合酮比色法检测各组细胞钙结节形成及钙含量;活性氧检测试剂盒(DCFH-DA探针)检测各组细胞活性氧阳性细胞数;蛋白免疫印迹法检测各组细胞中成骨转录因子Runx2的蛋白表达量。结果:钙化组活性氧的产生、钙结节、钙含量及Runx2蛋白表达量均显著高于对照组,钙化+过氧化氢组与钙化组相比,各项指标进一步升高,差异有统计学意义(P<0.05)。钙化+过氧化氢酶组活性氧产生量、钙结节、钙含量及Runx2蛋白表达量均低于钙化组和钙化+过氧化氢组,仍高于对照组,差异有统计学意义(P<0.05),但其中Runx2蛋白表达量与对照组相比则差异无统计学意义(P>0.05)。结论:氯化钙联合β-GP通过激活ROS-Runx2信号通路诱导大鼠VSMCs钙化形成。

Objective： To explore the effect of oxidative stress injury on the mechanism of vascular smooth muscle cell （VSMC） calciifcation induced by calcium and phosphorus in experimental rats. Methods： The VSMC calcification was induced by incubating the cells with calcium chloride （CaCl2） andβ-sodium glycerophosphate （β-GP） for 8 days, and the cells were divided into 4 groups： ① Control group, ② Calcification group,③ Calciifcation＋H2O2 group, ④ Calciifcation＋catalase group. The calcium nodule formation and calcium deposition in VSMC were detected by Alizarin red staining and o-cresolphthalein complexone method, the reactive oxygen species （ROS） was detected by DCFH-DA probe staining and the protein expression of Runx2 was examined by Western blot analysis. Results： Compared with Control group, Calciifcation group showed the higher ROS production, more calcium nodule and calcium deposition, higher Runx2 protein expression;while compared with Calciifcation group, the above indexes were even higher in Calciifcation＋H2O2 group, P〈0.05. The ROS production, calcium nodule, calcium deposition and Runx2 protein expression were lower in Calciifcation＋catalase group than those in Calciifcation group and Calciifcation＋H2O2 group, but still higher than that in Control group. The protein expression of Runx2 was similar between Calciifcation＋catalase group and Control group, P〉0.05. Conclusion： CaCl2 andβ-GP treatment may induce VSMC calciifcation via activating ROS-Runx2 signal pathway in experimental rats.