高磷对血管平滑肌钙化的影响及骨保护素干预作用

Effect of hyperphosphate on calcification of vascular smooth muscle cells and the intervention effect of osteoprotegerin

目的研究高磷对大鼠血管平滑肌细胞(RVSMC)钙化的影响和骨保护素(OPG)mRNA表达的影响,及骨保护素对高磷诱导RVSMC钙化的作用及其相关机制。方法用不同的试剂培养RVSMC,分别为正常磷组(Pi1.4mmol/L)、高磷组(Pi2.0mmol/L、Pi2.6mmol/L)、凋亡抑制组(Pi2.6mmol/L+ZVAD.FMK0.1μmol/L,Pi2.6mmol/L+ZVAD.FMK1.0μmol/L,Pi2.6mmol/L+ZVAD.FMK2.0μmol/L)、骨保护素组(Pi2.6mmol/L+OPG1μg/ml,Pi2.6mmol/L+OPG10μg/ml,Pi2.6mmol/L+OPG100μg/ml)。全自动生化仪比色法测定钙含量,BCA法测定蛋白含量,用蛋白含量标化钙含量。同时行VonKossa染色观察细胞钙化情况。用半定量PCR(RT-PCR)法检测RVSMCOPGmRNA的表达。流式细胞仪测定各组的细胞凋亡量。结果①培养3天,6天,9天时,高磷组(Pi2.0mmol/L,Pi2.6mmol/L)与Pi1.4mmol/L相比,RVSMC钙沉积较多(P<0.05);②培养第6天时,Pi2.6mmol/L+ZVAD.FMK0.1μmol/L组的钙沉积与Pi2.6mmol/L组相比无统计学差异,而Pi2.6mmol/L+ZVAD.FMK1.0μmol/L,Pi2.6mmol/L+ZVAD.FMK2.0μmol/L组与Pi2.6mmol/L组相比,RVSMC钙沉积较少(P<0.05)。Pi2.6mmol/L+OPG10μg/ml、Pi2.6mmol/L+OPG100μg/ml组与Pi2.6mmol/L组相比,细胞钙沉积较少(P<0.05)。Pi2.6mmol/L+OPG100μg/ml与Pi2.6mmol/L相比,细胞钙沉积明显降低(P<0.01);③VonKossa染色显示:Pi1.4mmol/L组钙化不明显;Pi2.6mmol/L组细胞有大量黑色颗粒沉积;而Pi2.6mmol/L+OPG100μg/ml组较少。培养第6天时,Pi2.6mmol/L组与Pi2.0mmol/L组;Pi2.0mmol/L,Pi1.4mmol/L相比,OPGmRNA表达较多(P<0.05)。培养第6天时,Pi2.6mmol/L与Pi1.4mmol/L相比,细胞凋亡量较多(P<0.05);Pi2.6mmol/L+OPG100μg/ml与Pi2.6mmol/L相比细胞凋亡量较少(P<0.05)。结论高磷可能通过诱导RVSMC凋亡而导致钙化,骨保护素对高磷诱导的RVSMC钙化有保护作用,此作用与其降低RVSMC凋亡有关。

Objective To study the effect of hyperphosphate on calcification and osteoprotegerin （OPG） mRNA expression in rat vascular smooth muscle cells （RVSMCs）, and the protection effect of OPG on RVSMCs calcification induced by hyperphosphate. Methods RVSMCs were cultured in normal phosphate medium, high phosphate medium, caspase inhibitor Z-VAD-FMK containing medium or OPG containing medium. Calcium content was quantified by the o-cresolphthalein complexone method, and protein content by the BCA method. RVSMCs calcification was visualized by Von Kossa staining. OPG mRNA was measured by semi-quantitative RT-PCR, and cell apoptosis was assayed by flow cytometry. Result （a） At the culture for 3, 6 and 9 days, RVSMCs calcification occurred more frequently in cells cultured in high phosphate medium than those in normal phosphate medium （P 〈 0.05）; （b） At the culture for 6 days, calcium deposition in RVSMCs was significantly inhibited in the cells cultured in 1.0μM/L and 2.0μM/L Z-VAD-FMK containing media （P 〈 0.05）, and in those cultured in 10μg/L and 100μg/L OPG containing media （P 〈 0.05）; （c） OPG mRNA increased in cells cultured in high phosphate medium, compared to those in normal phosphate medium; （d） Apoptosis and calcification of RVSMCs occurred frequently in those cultured in high phosphate medium. OPG significantly inhibited apoptosis in RVSMCs. Conclusion Hyperphosphate induces calcification of RVSMCs possibly through the induction of cell apoptosis. OPG protects RVSMCs from phosphate-induced calcification through inhibiting cell apoptosis.