尿毒症毒素对血管平滑肌细胞钙化影响的基础研究

Effect of Uremic Toxin on Calcification of Vascular Smooth Muscle Cells

目的:研究慢性肾衰竭尿毒症毒素高磷、高甲状旁腺激素各自对血管平滑肌细胞钙化的影响。方法:体外培养人胸主动脉血管平滑肌细胞(VSMC),分为对照组、高磷组(2.6mmoL/L)、高甲状旁腺激素(甲状旁腺素1-34,PTH1-34)a组(PTH1-34浓度10moL/L),高甲状旁腺激素(甲状旁腺素1-34,PTH1-34)b组(PTH1-34浓度10mo L/L),培养14d,茜素红染色后观察细胞钙盐沉积情况;实时定量PCR和western blotting检测骨形态发生蛋白2(BMP-2)及Runt相关转录因子2(Runx-2)的基因和蛋白表达。应用细胞内钙含量试剂盒测定各组细胞内钙含量。结果:茜素红染色发现高磷组血管平滑肌细胞钙化,western blotting检测表达钙化蛋白BMP-2和Runx-2明显增高。茜素红染色未发现高甲状旁腺激素组培养的血管平滑肌细胞钙化,且实时定量PCR和western blotting检测高甲状旁腺激素组血管平滑肌细胞BMP-2及Runx-2表达较对照组无增加。高磷组细胞胞内钙含量明显高于对照组,高甲状腺素a组和b组细胞内钙含量与正常对照组细胞无差异(P>0.05)。结论:单纯高磷诱导的血管平滑肌细胞钙化,而单纯的高甲状腺素刺激14d不能使血管平滑肌细胞钙化。

Objective:To study the respective effects of high phosphorus or high parathyroid hormone levels on smooth muscle cell calcification.Methods:Human thoracic aortic vascular smooth muscle cells(VSMC)were cultured in vitro for 14 days and divided into control group,high phosphorus group(2.6 mmol/L),high parathyroid hormone(parathyroid hormone 1-34,PTH1-34)group a(PTH1-34 concentration 10mol/L),high parathyroid hormone(parathyroid hormone 1-34,PTH1-34)group b(PTH1-34 concentration 10mol/L),and observed in respect of cellular calcium salt deposition after alizarin red staining;real-time quantitative PCR and western blotting were used to detect the gene and protein expression of bone morphogenetic protein 2(BMP-2)and Runt-related transcription factor 2(Runx-2).The intracellular calcium content of each group was determined by applying the intracellular calcium content kit.Resulst:Calcification of vascular smooth muscle cells in the high phosphorus group was detected by alizarin red staining,and expression of calcification proteins BMP-2 and Runx-2 was significantly increased by western blotting.Calcification of vascular smooth muscle cells cultured in the high parathyroid hormone group was not detected by alizarin red staining,and BMP-2 and Runx-2 expression in vascular smooth muscle cells in the high parathyroid hormone group was not increased by real-time quantitative PCR and western blotting compared with the control group.expression did not increase compared with the control group.The intracellular calcium content of cells in the high phosphorus group was significantly higher than that of the control group,and the intracellular calcium content of cells in the high parathyroid hormone a and b groups did not differ from that of normal control cells(P>0.05).Conclusion:The calcification of VSMCs was induced merely by high phosphorus,However,PTH stimulation for 14 days failed to calcify vascular smooth muscle cells.