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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。
AIM: To investigate the role of bone morphogenetic protein 2 (BMP-2) in the calcification of rat thoracic aorta rings induced by high phosphorus. Methods: Thoracic aortic rings of 8 ~ 10 weeks old male Sprague - Dawley rats were cultured in vitro. The vascular rings were randomly divided into two groups according to random number table: normal control group and high phosphorus group. After cultured for 7d and 14d, von Kossa staining and o-cresolphthalein complex colorimetry were used to detect the calcification of the thoracic aorta rings in rats. The expression of BMP-2 in the vascular rings was detected by immunohistochemistry. In vitro, primary rat aorta smooth muscle cells (VSMCs) were cultured with tissue-attached method. The vascular smooth muscle cell calcification model was prepared by 10mmol / Lβ-glycerophosphate. Cells were randomly divided into 2 groups: normal control group, - Glycerophosphate). Alizarin red staining and o-cresolphthalein complex ketone colorimetric assay was used to detect cell calcification. RT-PCR and Western blot were used to detect the expression of BMP-2mRNA and protein at different time points -2 protein expression. Results: Compared with the normal control group, the content of calcium in high-phosphorus group increased significantly (P <0.05) after 7d and 14d of culture. Compared with the calcium content of vascular ring in 7d high phosphorus group, Content was significantly increased (P <0.05). Immunohistochemistry results showed that compared with the normal control group, the expression of BMP-2 in high phosphorus group increased (P <0.05), and the calcium content in vascular ring in 14d high phosphorus group increased significantly compared with 7d high phosphorus group (P <0.05) . Compared with the normal control group, calcium deposition and calcium content in the high phosphorus group were significantly increased (P <0.05) after 7 and 14 days of cell culture. Compared with the normal control group, RTPCR and Western Blot showed that BMP-2 mRNA And protein expression increased. Further dynamic observation of BMP-2 protein expression changes, the results showed that BMP-2 protein expression in normal control group and high phosphorus group increased with time (P <0.05). Conclusion: BMP-2 may be involved in the development of vascular calcification induced by high phosphorus.