中国血液净化

›› 2007, Vol. 6 ›› Issue (5): 267-271.

• 基础研究 • 上一篇    下一篇

高钙、高磷诱导的大鼠血管平滑肌细胞钙化过程中骨桥蛋白表达的动态变化

王 英1 王 梅2

  

  1. 1. 100034 北京，北京大学第一医院肾内科 2. 100044 北京，北京大学人民医院肾内科
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2007-05-12 发布日期:2007-05-12

  • Received:1900-01-01 Revised:1900-01-01 Online:2007-05-12 Published:2007-05-12

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摘要： 目的 观察大鼠血管平滑肌细胞(VSMCs)在高钙、高磷条件下骨桥蛋白(OPN)表达的动态变化及其分布特点，探讨其与血管钙化的关系。 方法 应用大鼠原代VSMCs进行体外实验，细胞生长至80%融合后更换不同培养基进行培养。根据培养基中钙磷浓度不同分为3组：对照组、高钙组和高磷组。更换培养基当天定义为D0。应用原子分光光度计检测细胞内钙含量。应用免疫组化染色观察各组第3、6、10和17天OPN的在细胞中的表达；同时收集第4、8、12天各组细胞，提取总蛋白，Western blot方法检测OPN表达。最后应用电镜、免疫电镜观察OPN在VSMCs的分布。结果 对照组未观察到钙盐沉积，VSMCs钙含量在10天内无明显变化，高钙组VSMCs钙含量D2有明显增加，D8、D10进一步增加；高磷组钙含量D8、D10明显增加，与D0比较差异均有显著性(P＜0.01)。在D0-D10，高钙组、高磷组VSMCsOPN的表达逐渐增强。但这以后，无论是D12 Western blot结果，还是D17免疫组化结果均显示OPN表达下调。对照组OPN表达一直很弱，且随时间变化无明显性。D10高钙组和高磷组VSMCs在电镜下可见细胞外基质沉积，为丰富的胶原纤维，其间可见直径100～200nm的小泡。高密度电子致密物主要呈颗粒样沿胶原纤维沉积；也可见于小泡的泡壁。免疫电镜显示，高钙组和高磷组VSMCs胞浆中均可见较多与OPN相结合的胶体金颗粒，而在细胞外基质中仅偶见。结论 大鼠VSMCs在高钙和高磷条件下培养发生钙化过程中， OPN表达呈先增强再减弱的变化；对照组OPN的表达一直在一个很低的水平。免疫电镜显示OPN主要表达于细胞胞浆中，细胞外基质分布很少。

关键词: 血管平滑肌细胞, 高钙, 高磷, 骨桥蛋白

Abstract: Objective To investigate the change of osteopontin (OPN) expression and its distribution in the calcification of rat vascular smooth muscle cells (VSMCs) induced by high phosphate and high calcium media. Methods Primary VSMCs from rat aortic media were cultured in 3 media: normal calcium and phosphate medium (control group, 1.4mmol/L phosphate and 2.0mmol/L Ca2+), high calcium medium (high calcium group, 1.4mmol/L phosphate and 2.8mmol/L Ca2+) and high phosphate medium (high phosphate group, 2.4 mmol/L phosphate and 2.0mmol/L Ca2+). Calcium deposition in the cells was measured by atomic absorptiometry. Change of OPN expression in the calcification process was detected by immunohistochemistry and immunoblotting. Ultrastructural changes of the calcified cells at the day 10 in high calcium and high phosphate groups were observed by transmission electron microscope. OPN distribution in the cells was also examined by immunoelectron microscopy. Results After 10 days of the treatment, calcium deposition measured by atomic absorptiometry was significantly increased in both high calcium group and high phosphate group (P＜0.01). VSMCs in these two groups were observed to increasingly express OPN from the day 0 to day 10. After day 10, OPN expression was down-regulated. In control group, OPN expression remained unchanged at a very low level during the culture. Transmission electron microscopy showed that collagen fibers and vesicles of 100-200nm in diameter among the collagen fibers were found in the extracellular matrix in high calcium group and high phosphate group at the day 10. Electron-dense granular deposits situated along the collagen fibers and on the matrix vesicles. After immunocytochemical labeling for OPN antibody, gold particles were located in cell plasma, but seldom found in the extracellular matrix. Conclusion During calcification process of VSMCs induced by high phosphate and high calcium media, OPN expression was up-regulated first and then down-regulated. In control group, OPN expression remained at a low level. By immunoelectron microscopy, OPN was found in cell plasma but little in extracellular matrix.

Key words: High calcium, High phosphate, Osteopontin

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