高磷对牛血管平滑肌细胞钙沉积和骨钙素影响的实验研究

Hyperphosphate to Evoke Calcium Deposition of Vascular Smooth Muscle Cell and Expression of Osteocalcin

目的:观察不同磷浓度(Pi)对培养的牛主动脉血管平滑肌细胞钙沉积和骨钙素水平的直接影响,并探讨高磷血症诱导血管钙化的机制。方法:用不同Pi的培养液体外培养牛主动脉平滑肌细胞,用甲o-酚酞络合酮方法测定钙含量,放射免疫法测定培养上清液中骨钙素的浓度,BCA(Bicinchoninicacid)法测定蛋白含量,用蛋白含量标化钙沉积、骨钙素的浓度。结果:与相当于正常血磷(1.5mmol/L)的培养基中钙沉积相比,在相当于高磷血症(2.0mmol/L)的培养基中,钙沉积显著增加犤培养6天,Pi2.0mmol/L组为(77.19±11.69)μg/mg蛋白;Pi1.5mmol/L组为(25.77±1.75)μg/mg蛋白,P<0.01犦,呈时间和剂量依赖性。vonKossa钙染色,Pi2.0mmol/L组培养的平滑肌细胞中见大量的黑色颗粒沉积,而Pi1.5mmol/L组中很少。同样,与Pi1.5mmol/L组相比,Pi2.0mmol/L组培养上清液中骨钙素的水平明显增高犤Pi2.0mmol/L组为(1.50×10-2±2.60×10-3)ng/μg蛋白,Pi1.5mmol/L组为(2.98×10-3±8.38×10-4)ng/μg蛋白,P<0.001犦。结论:高磷可直接刺激平滑肌细胞的钙沉积和骨钙素产生增加,促进血管钙化的发生。高磷血症是血管钙化发生的独立因素。

Objective:To evaluate the effect of different concentrations of phosphorus on calcium deposition and osteocalcin level in bovine aortic smooth muscle cell cultures and investigate the mechanism of hyperphosphatemia to evoke calcification of vascular smooth muscle cell.Methods:The culture of bovine aortic smooth muscle cells(BASMC) were achieved.Calcium deposition and the expression of osteocalcin in two different concentrations of phosphate (1.5 mmol/L and 2.0 mmol/L) were determined by o-cresolphthalein complexone and radioimmunity methods respectively.Results:After six or nine days of BASMC culture ,the calcium deposition in Pi 2.0 mmol/L group was more than that in Pi 1.5 mmol/L group [(77.19±11.69)μg/mg protein versus (25.77±1.75)μg/mg protein,P< 0.01 and (125.39±16.68)μg/mg protein versus (29.05±2.64)μg/mg protein,P< 0.01 respectively].The calcium deposition was dependent on time and dosage of phosphate treatment. After 72 h culture the osteocalcin in Pi 2.0 mmol/L group was more than that in Pi 1.5 mmol/L group [(1.50×10-2±2.60×10-3) ng/μg protein versus (2.98×10-3±8.38×10-4)ng/μg protein,P< 0.001].Conclusion:Hyperphosphate may directly promote calcium deposition and the expression of osteocalcin of BASMCs and,it may be a new explanation of the phenomenon on vascular calcification under hyperphosphatemic conditions. Hyperphosphatemia is an independent factor to stimulate vascular calcification.