摘要
目的探讨镁离子对大鼠血管平滑肌细胞（VSMCs）钙化相关因子表达水平的影响。方法选择5周龄健康雄性SD大鼠6只。以组织贴壁法进行原代VSMCs培养,至3~5代时用于细胞实验。将VSMCs采用随机数字表法分为正常对照组、高磷组（加入10 mmol/Lβ-甘油磷酸）、镁干预组〔加入10 mmol/Lβ-甘油磷酸＋3 mmol/L硫酸镁（Mg SO4）〕、2-氨基乙氧基二苯基硼酸（2-APB）干预组（10 mmol/Lβ-甘油磷酸＋3 mmol/L Mg SO4＋10-4μmol/L 2-APB）。采用邻甲酚酞络合酮比色法检测细胞培养14 d后钙化情况,酶联免疫吸附法检测细胞培养14 d后碱性磷酸酶（ALP）活性,反转录聚合酶链反应（RT-PCR）检测0、3、6、10、14 d时细胞内基质G蛋白（MGP）mRNA和骨桥蛋白（OPN）mRNA的表达水平。结果细胞培养14 d后,4组钙水平比较,差异有统计学意义（F=409.800,P〈0.05）;高磷组、2-APB干预组钙水平高于正常对照组和镁干预组（P〈0.05）。4组ALP活性比较,差异有统计学意义（F=490.901,P〈0.05）;高磷组、2-APB干预组ALP活性高于正常对照组和镁干预组（P〈0.05）。培养14 d后,4组MGP mRNA和OPN mRNA的表达水平比较,差异有统计学意义（F=1 279.905、1 157.247,P〈0.05）;镁干预组MGP mRNA和OPN mRNA的表达水平高于高磷组及2-APB干预组（P〈0.05）。动态观察MGP mRNA和OPN mRNA的表达变化,结果显示在培养第3天时,高磷组MGP mRNA和OPN mRNA表达水平均降低（P〈0.05）,并随着时间的延长呈下降趋势;镁干预组MGP mRNA和OPN mRNA表达水平均升高（P〈0.05）,并随着时间的延长呈上升趋势。结论高浓度镁离子在抑制VSMCs钙化过程中可上调MGP及OPN的表达,并呈一定时间依赖性,为临床进一步研究提供了参考依据。
Objective To investigate the effect of magnesium on the expression of factors related to calcification in rat vascular smooth muscle cells（ VSMCs）. Methods A total of 6 healthy SD rats aged 5 weeks were selected. VSMCs were primarily cultured in vitro using tissue block adhering wall method, and the third to fifth generation was used for cell experiment. The VSMCs were randomly divided into control group,high phosphorus group（ 10 mmol / L β- glycerophosphate）,magnesium intervention group（ 10 mmol / L β- glycerophosphate and 3 mmol / L Mg SO4）,2- APB intervention group（ 10 mmol/L β- glycerophosphate,3 mmol / L Mg SO4 and 10- 4μmol /L 2- APB）. After culturing for 14 days,calcification was detected with OCPC method and the activity of alkaline phosphatase was detected with enzyme linked immunosorbent assay（ ELISA）method. The expression of matrix gla protein（ MGP） mRNA and osteopontin（ OPN） mRNA on 0,3,6,10 and 14 days were detected by RT- PCR method. Results After culturing for 14 days,the four groups showed statistically significant differences in calcification level（ F = 409. 800,P〈 0. 05）,with the high phosphorus group and 2- APB intervention group significantly higher than the control group and magnesium intervention group（ P〈 0. 05）. The four groups showed statistically significant differences in ALP activity（ F = 490. 901,P〈 0. 05）,with the high phosphorus group and 2- APB intervention group significantly higher than the control group and magnesium intervention group（ P〈 0. 05）. The four groups showed statistically significant differences in the expression of MGP mRNA and OPN mRNA（ F = 1 279. 905,1 157. 247,P〈 0. 05）,with the magnesium intervention group significantly higher than the phosphorus group and 2- APB intervention group（ P〈 0. 05）. Dynamic observation showed that the expression of MGP mRNA and OPN mRNA decreased after culturing for three days（ P〈 0. 05） and the decrease was in a time dependent manner. However,the expression of MGP mRNA and OPN mRNA in magnesium intervention group increased（ P〈 0. 05） in a time dependent manner. Conclusion Magnesium may up- regulate the expression of MGP and OPN in a time dependent manner in inhibiting the calcification of VSMCs,providing a reference for further clinical study.
出处
《中国全科医学》
CAS
CSCD
北大核心
2015年第6期665-668,共4页
Chinese General Practice
基金
河北省自然科学基金资助项目(H2012206157)
作者简介
通信作者：徐金升，050011河北省石家庄市，河北医科大学第四医院肾内科；E—mail：xjs5766@126．com
