论文部分内容阅读
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、20μmol/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 inhibitory effect of resveratrol (Res) on vascular calcification and to investigate the effects of different concentrations of Res on the osteogenic transformation of human umbilical arterial smooth muscle cells (hVSMC) induced by high calcium and high phosphorus. Methods hVSMC cells were cultured in vitro and identified by morphological and α-SMA labeling. Osteogenic transformation of hVSMC was induced by medium of high calcium and high phosphorus (2.5 mmol / L of calcium and 3.0 mmol / L of phosphorus), and osteogenic transformation was identified by the method of alizarin. The normal group was treated with different concentrations of Res (5, 10, 20μmol / L). After cultured for 12 days, the cells in each group were assayed for calcium The expressions of bone-specific alkaline phosphatase (BAP), bone morphogenetic protein (BMP-2) and osteopontin (OPN) in hVSMC were detected by real-time fluorescence quantitative PCR. Western blotting and immunofluorescence staining Methods The expression of BAP, BMP-2 and OPN protein were detected. Results Compared with the control group, the content of calcium in the positive control group increased about 5-fold and the expression of α-SMA protein decreased, while the expression of BAP, BMP-2, OPN mRNA and protein in osteoblast-like cells increased significantly High (P <0.01). After intervention with different concentrations of Res, the intracellular calcium decreased, while the levels of BAP, BMP-2 and OPN mRNA decreased (P <0.05, 0.01); the protein expressions of BMP-2 and OPN were significantly decreased Res concentration was negatively correlated. Conclusion hVSMC undergoes osteogenic transformation under the condition of high calcium and high phosphorus, while Res inhibits the progression of the process and is positively correlated with its concentration.