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目的 观察高磷对培养的牛主动脉平滑肌细胞骨钙素(OC)mRNA表达和钙沉积的影响,探讨高磷是否为促进血管钙化的独立因素。方法 用不同磷浓度[正常组(Pi 1.5 mmol/L)、高磷组(2.0 mmol/L)]的培养液,培养牛主动脉平滑肌细胞,观察72 h骨钙素的表达,以及不同时间(第3、6、9天)血管平滑肌细胞的钙沉积。上清液中骨钙素浓度用放射免疫法测定。RT-PCR观察骨钙素mRNA的表达。平滑肌细胞钙含量用甲O-酚酞络合酮方法测定。BCA法测定蛋白含量,用蛋白含量标化骨钙素浓度和钙含量。结果 3d后与正常磷组相比,高磷组上清液骨钙素水平明显增高[高磷组(15.03±2.60)pg/μg蛋白比正常磷组(2.98±0.84)pg/μg蛋白,P<0.001];高磷组平滑肌细胞骨钙素mRNA的表达也显著增加(OC/GAPDH:1.91±0.13比0.75±0.04,P<0.001)。高磷组钙沉积显著增加[培养第6天,高磷组(77.19±11.69)μg/mg蛋白比正常磷组(25.77±1.75)μg/mg蛋白,P<0.01],呈时间和剂量依赖性。von Kossa钙染色,高磷组平滑肌细胞中见大量的黑色颗粒沉积。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,促进血管平滑肌细胞钙化。高磷血症是促发血管钙化的独立因素。
Objective To observe the effect of high phosphorus on osteocalcin (OC) mRNA expression and calcium deposition in cultured bovine aortic smooth muscle cells (VSMCs), and to explore whether high phosphorus is an independent factor promoting vascular calcification. Methods Bovine aortic smooth muscle cells were cultured in different concentrations of phosphorus (Pi 1.5 mmol / L, 2.0 mmol / L) and the expression of osteocalcin at 72 hours was observed. 3rd, 6th and 9th days) calcium deposition in vascular smooth muscle cells. The concentration of osteocalcin in supernatant was determined by radioimmunoassay. The expression of osteocalcin mRNA was observed by RT-PCR. The calcium content of smooth muscle cells was measured by the method of O-phenolphthalein complex ketone. BCA method for the determination of protein content, protein content of the standardization of osteocalcin concentration and calcium content. Results Compared with normal group, the level of osteocalcin in hyperphosphorus group was significantly higher than that in normal group after 3 days (15.03 ± 2.60 pg / μg protein vs 2.98 ± 0.84 pg / μg protein, P <0.001]. The expression of osteocalcin mRNA in smooth muscle cells of high phosphorus group also increased significantly (OC / GAPDH: 1.91 ± 0.13 vs 0.75 ± 0.04, P <0.001). The calcium deposition in high phosphorus group increased significantly (P <0.01) on the 6th day, compared with the control group (77.19 ± 11.69 μg / mg protein, 25.77 ± 1.75 μg / mg protein, P <0.01) . von Kossa calcium staining, a large number of black particles deposition in the high phosphorus group of smooth muscle cells. Conclusion High phosphorus can directly stimulate the calcium deposition and osteocalcin production of smooth muscle cells and promote the calcification of vascular smooth muscle cells. Hyperphosphatemia is an independent factor to promote vascular calcification.