目的 研究白藜芦醇（resveratrol，Res）对血管钙化的抑制作用，观察不同浓度的Res对高钙高磷诱导的人脐动脉血管平滑肌细胞（hVSMC）成骨样转化的影响。方法 体外培养hVSMC细胞，通过形态学和α-SMA标记进行鉴定。用高钙高磷（钙2.5 mmol/L、磷3.0 mmol/L）培养基诱导hVSMC成骨样转化，通过茜红素染色法鉴定成骨样转化。以普通培养hVSMC作为对照组，加高钙高磷为阳性对照组，实验组为阳性对照加不同浓度的Res（5、10、20 μmol/L）干预，培养12 d后，检测各组细胞含钙量，实时荧光定量PCR检测hVSMC成骨样转化相关基因骨特异性碱性磷酸酶（BAP）、骨形态蛋白（BMP-2）、骨桥蛋白（OPN）的表达水平；Western blotting和免疫荧光标记方法检测BAP、BMP-2、OPN蛋白的表达。结果 与对照组比较，阳性对照组细胞内含钙量明显升高约5倍，α-SMA蛋白表达下降，而成骨样细胞特异性标记BAP、BMP-2、OPN mRNA和蛋白质表达均显著升高（P<0.01）。不同浓度Res干预后后，细胞内含钙量下降，BAP、BMP-2、OPN mRNA水平降低（P<0.05、0.01）；BMP-2及OPN的蛋白表达显著降低（P<0.01），并与Res浓度呈负相关。结论 hVSMC在高钙高磷条件下发生成骨样转化，而Res抑制此过程的进展并与其浓度呈正相关。

Objective To investigate the inhibition of resvertrol (Res) on angiosteosis, and to observe the effects of Res at different concentration on osteogenic conversion of human umbilical artery vascular smooth muscle cells (hVSMCs) induced by elevated calcium (Ca²⁺) and phosphate (P) culture. Methods The hVSMCs were cultured in vitro, and identified by morphology and α-smooth muscle actin (α-SMA) antibody. High calcium (2.5 mmol/L) and high phosphate (3.0 mmol/L) culture was used to induce the osteogenic differentiation of hVSMCs. The calcification was confirmed by Alizarin red staining. Cells were divided into five groups such as normal control (NC, normal concentration of Ca²⁺ and P), positive control (PC, high Ca²⁺ and high P), and three Res (5, 10, and 20 μmol/L) groups. Calcium deposition in cells was measured by Arsenazo III after 12 d. Real-time PCR and Western blotting were used to detect mRNA and protein expression of BAP, BMP2, and OPN. Results Compared to NC group, calcium deposition in the cells dramatically increased by five times in PC group, the protein expression of α-SMA decreased, and the mRNA and protein expression of BAP, BMP-2, and OPN obviously increased (P < 0.05). Res at different concentration could reduce the cellular Ca²⁺. The levels of BAP, BMP2, and OPN were highest in PC group than NC and three Res groups. Furthermore, the expression of BMP2 and OPN decreased with increasing Res concentration. Conclusion Res prevents the process of the osteogenic differentiation of hVSMCs induced by high calcium and high phosphate. The degree of osteogenic differentiation is falling with increasing Res concentration.

浙江省自然科学基金资助项目（LY12H05004）；温州市科技局社会发展科学研究项目（Y20100224）；浙江省科技厅项目（2010C33094）