摘要
目的观察不同浓度磷对体外培养的牛主动脉平滑肌细胞钙沉积及骨钙素表达的影响,同时观察膦甲酸钠对高磷诱导该细胞钙化的干预作用。方法体外培养牛主动脉平滑肌细胞,观察不同磷浓度(Pi 1.5、2.0 mmol.L-1)、不同培养时间(3、6、9 d)血管平滑肌细胞的钙沉积,以及不同磷浓度(Pi 1.5、2.0、2.5 mmol.L-1)培养血管平滑肌细胞72 h骨钙素的表达。同时观察不同浓度膦甲酸钠对高磷(Pi 2.0 mmol.L-1)诱导的钙沉积和骨钙素增加的抑制作用。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法测定蛋白含量,用蛋白含量标化钙含量与骨钙素浓度;RT-PCR测定骨钙素mRNA的表达。结果在相当于正常血磷浓度(1.5 mmol.L-1)的培养基中,平滑肌细胞钙沉积量小;而在相当于高磷血症(2.0 mmo.lL-1)的培养基中,钙沉积明显增加[培养6 d,(77.187±11.692)vs(25.768±1.750)μg.(mg蛋白)-1,P<0.01],并呈时间和剂量依赖性。与Pi 1.5 mmo.lL-1组相比,Pi 2.0 mmol.L-1组培养上清液中骨钙素的水平明显增高[(1.503×10-2±2.601×10-3)vs(2.981×10-3±8.382×10-4)ng.(μg蛋白)-1,P<0.001];Pi 2.0 mmol.L-1组平滑肌细胞骨钙素mRNA的表达也明显增加(OC/GAPDH,1.906±0.132vs0.748±0.036 6,P<0.001)。膦甲酸钠能有效地抑制钙沉积[培养6 d,Pi 2.0 mmol.L-1+PFA 1.0 mmol.L-1组vsPi 2.0 mmol.L-1组,(37.729±5.899)vs(77.187±11.692)μg.(mg蛋白)-1,P<0.001]和上清液中骨钙素的表达[(4.529×10-3±1.250×10-3)vs(1.503×10-2±2.601×10-3)ng.(μg蛋白)-1,P<0.001]及平滑肌细胞骨钙素mRNA的表达(OC/GAPDH,0.642±0.092vs1.885±0.165,P<0.01)。结论高磷能直接促进血管平滑肌细胞钙沉积和骨钙素表达的增加,说明高磷血症可能是促进血管钙化的独立危险因素;膦甲酸钠能有效地抑制高磷诱导的血管平滑肌细胞钙沉积和骨钙素表达的增加,可以作为一种新的防治高磷诱导血管钙化的药物。
Objective To evaluate the effects of different concentrations of phosphate on calcium deposition and osteocalcin level in bovine aortic smooth muscle cell cultures and observe the effects of phosphonoformic acid （ PFA ） in different concentrations on vascular calcification induced by elevated phosphate. Methods Bovine aortic smooth muscle cells （BASMC）were cultured. Calcium deposition and the expression of osteocalcin in different concentrations of phosphate （ 1.5 and 2.0 mmol· L^-1） and PFA were determined by ocresolphthalein complexone and radioimmunity methods respectively, osteocalcin mRNA expression were determined by RT-PCR. Results After six days of BASMC culture, the calcium deposition in Pi 2.0mmol· L^-1 group was more than that in Pi 1.5 mmol· L^-1 group[ （77. 187 ± 11. 692）vs（25. 768 ± 1. 750） μg·（ mg protein）^-1 ,P〈0.01 ]. The calcium deposition was dependent on time and dosage of phosphate treatment. After 72 h culture, the osteocalcin in Pi 2.0mmol· L^-1 group was more than that in Pi 1.5mmol· L^-1 group [ （1.503×10^-2±2. 601 × 10^-3） vs（2. 981 × 10-3 ±8. 382 × 10^-4） ng·（μg protein）^-1,P〈0. 001） ,the same as osteocalcin mRNA expression （ OC/GAPDH, 1. 906 ± 0. 132 vs0. 748 ± 0. 036 6, P 〈 0. 001 ）. PFA decreased ciacium deposition [ Pi 2.0 mmol· L^-1 ± PFA1.0mmol· L^-1 group vs Pi 2.0 mmol· L^-1 group, （37. 729 ± 5. 899 ） vs （ 77. 187 ± 11. 692 ）μg·（mg protein）^-1,P〈 0. 001] and osteocalcin expression [ （4.529 × 10^-3± 1. 250 × 10^-3 ） vs （ 1. 503 × 10^-2 ± 2. 601 × 10^-3 ） ng· （ μg protein）^-5, p 〈 0. 001 ] statistically, as well as osteocalcin mRNA expression （ OC/GAPDH,0. 642 ± 0. 092 vs 1. 885 ± 0. 165, P 〈 0.01 ）. Conclusion Hyperphosphate may directly promote calcium deposition and the osteocalcin expression of BASMCs,it may be a new explanation of the phenomenon on vascular calcification under hyperphosphatemic conditions. Hyperphosphatemia is an independent factor to stimula
出处
《现代医学》
2008年第2期73-77,共5页
Modern Medical Journal
关键词
血管平滑肌细胞
磷酸盐
钙化
骨钙素
膦甲酸钠
vascular smooth muscle cell
phosphate
calcification
osteocalcin
phosphonoformic acid
