中国血液净化

›› 2008, Vol. 7 ›› Issue (2): 85-89.

• 基础研究 • 上一篇    下一篇

高钙、高磷对体外培养的大鼠血管平滑肌细胞钙化的作用

王 英　 王 梅   

  1. 北京大学第一医院肾内科
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2008-02-12 发布日期:2008-02-12
  • 通讯作者: 王梅

The effect of high calcium and high phosphate on calcification of rat VSMCs

WANG Ying, WANG Mei   

  • Received:1900-01-01 Revised:1900-01-01 Online:2008-02-12 Published:2008-02-12
  • Contact: WANG Mei

RichHTML

0

PDF

429

摘要： 【摘要】目的 研究高钙、高磷对体外培养的原代大鼠血管平滑肌细胞(VSMCs)钙沉积以及VSMCs、成骨细胞标志蛋白表达的情况。方法 应用大鼠原代血管平滑肌细胞进行体外实验，根据培养基中钙、磷浓度不同分为三组：正常钙、磷组(对照组)、高钙组和高磷组。以更换培养液当天为D0。在培养的10天内观察细胞的形态学变化，应用原子分光光度计检测细胞内钙含量。应用免疫组化染色和Western blot方法观察VSMCs及成骨细胞标志蛋白的表达。结果 对照组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D8钙含量有明显增加，D10进一步增加，与D0比较均有显著差异(P＜0.01)。Western blot显示高钙组、高磷组平滑肌肌动蛋白α(α-SMA)表达在D12较D0分别下降52%和57%，而对照组无明显变化。D17α-SMA免疫组化染色此两组细胞胞浆中几乎无表达。免疫组化染色显示，高钙组和高磷组VSMCs在培养的10天内，成骨细胞标志蛋白核心结合因子α1(Cbfα1)、I型胶原(ColI)和骨桥蛋白(OPN)表达并逐渐增强，对照组仅有微弱表达，且随时间无明显变化。Western blot显示高钙组和高磷组D8 OPN的表达明显高于对照组，为对照组的2.6倍。结论 大鼠VSMCs在高钙或高磷条件下出现钙盐沉积。在此过程中，VSMCs发生细胞表型转化。高钙、高磷可能通过使血管平滑肌细胞转化为成骨样细胞导致钙化发生。

关键词: 血管平滑肌细胞, 高钙, 高磷, 血管钙化, 成骨细胞

Abstract: 【Abstract】 Objective To investigate the effect of high phosphate and high calcium on calcification of rat vascular smooth muscle cells (VSMCs) and on the expression of specific VSMC and osteoblast markers. Methods VSMCs from explants of rat aortic media were cultured. Cells were divided into three groups: normal calcium and phosphate group (control group, culture medium containing 1.4mmol/L phosphate and 2.0mmol/L calcium), high calcium group (culture medium containing 1.4mmol/L phosphate and 2.8mmol/L calcium) and high phosphate group (culture medium containing 2.4mmol/L phosphate and 2.0mmol/L calcium). The first day of cells cultured in a specific medium was defined as day 0. Calcium deposition in cells was measured by atomic absorptiometry. The expression of smooth muscle lineage marker α-SMA and osteoblast markers Cbf-α1, collagen I and osteopontin (OPN) were detected by immunohistochemistry and western blotting. Results After culture for 10 days, calcium deposition increased significantly in high phosphate group and high calcium group (P＜0.01). In contrast, calcium deposition was trivial in control group. In response to high calcium or high phosphate media, rat VSMCs lost the expression of VSMC marker α-SMA and expressed higher level of Cbf-α1, collagen I and OPN. Conclusion High concentration of calcium and phosphate may directly stimulate rat VSMCs changing their phenotype that predisposes the cells to be calcified.

Key words: High calcium, High phosphate, Vascular calcification, Osteoblast