高磷对大鼠胸主动脉血管环中骨形成蛋白2表达的影响

Effect of high phosphorus on the expression of bone morphogenetic protein 2 in rat aortic rings

目的:探讨骨形成蛋白-2(Bone morphogenetic protein 2,BMP-2)在高磷诱导的大鼠胸主动脉血管环钙化中的作用。方法:选取8~10周龄健康雄性SD大鼠,体外培养大鼠胸主动脉血管环,血管环按随机数字表法随机分为2组:正常对照组和高磷组。血管环培养7d和14d后,采用von Kossa染色及邻甲酚酞络合酮比色法检测大鼠胸主动脉血管环钙化情况;免疫组织化学方法检测血管环BMP-2的表达。体外采用组织块贴壁法培养原代大鼠胸主动脉平滑肌细胞,利用10mmol/Lβ-甘油磷酸制备血管平滑肌细胞钙化模型,细胞随机分为2组:正常对照组、高磷组(10mmol/Lβ-甘油磷酸)。采用茜素红染色及邻甲酚酞络合酮比色法检测细胞钙化情况,RT-PCR和Western blot方法检测细胞培养7d和14d的BMP-2mRNA及蛋白表达,并观察短时间内不同时间点BMP-2蛋白表达情况。结果:体外血管环培养7d、14d后,与正常对照组相比,高磷组钙含量明显增加(P<0.05);与7d高磷组血管环钙含量相比,14d高磷组血管环钙含量明显增加(P<0.05)。免疫组织化学结果显示,与正常对照组相比,高磷组BMP-2表达增加(P<0.05);与7d高磷组相比,14d高磷组血管环钙含量明显增加(P<0.05)。细胞培养7d、14d后,与正常对照组相比,高磷组钙盐沉积及钙含量明显增高(P<0.05);RTPCR和Western Blot显示,与正常对照组相比,高磷组BMP-2mRNA及蛋白表达增加。进一步动态观察BMP-2蛋白的表达变化,结果显示正常对照组及高磷组BMP-2蛋白表达随时间延长呈增强趋势(P<0.05)。结论:BMP-2可能参与了高磷诱导的血管钙化的发生发展。

Objective：To investigate the role of bone morphogenetic protein 2（BMP-2） in the vascular calcification of the thoracic aorta in rats induced by high phosphorus.Method：The 8~10 weeks old healthy male SD rats were selected and cultured in vitro.The vascular rings were randomly divided into 2 groups：normal control group and high phosphorus group.Vascular rings cultured 7 days and 14 days,using von Kossa staining and ocresolphthalein complexone colorimetry to detect rat thoracic aorta calcification.The expression of vascular ring BMP-2 was detected by immunohistochemistry method.In vitro by tissue adherent cultured primary rat aortic smooth muscle cells and calcification of vascular smooth muscle cells model were prepared by 10 mmol/L of β-glycerophosphate system,cells were divided for 2 groups：normal control group,high phosphorus（10 mmol/L beta glycerophosphate）group.Result：In vitro vascular ring were cultured for 7days and 14 days.Compared with the normal control group,the content of calcium was significantly increased in high phosphorus group（P〈0.05）.Compared with 7 days of high phosphorus group of vascular ring calcium content,14 days high phosphorus group of vascular ring calcium content was significantly increased（P〈0.05）.Immunohistochemistry results showed that compared with the normal control group,and the expression of BMP-2 was increased in high phosphorus group（P〈0.05）.Compared with the 7 high phosphorus group,the calcium content of ring blood vessels was increased in 14 days of high phosphorus group（P〈0.05）.After 14 days and 7 days,the calcium salt deposition and the calcium content of the high phosphorus group were significantly increased compared with the normal control group（P〈0.05）.RT-PCR and Blot Western showed that,compared with the normal control group,the expression of BMP-2 mRNA and protein in high phosphorus group increased.We further observed the change of BMP-2 protein expression.The results showed that the expression of BMP-2 protein was increased with the time in the normal control group and the high phosphorus group（P〈0.05）.Conclusion：BMP-2 may be involved in the occurrence and development of vascular calcification induced by high phosphorus.