镁通过L型钙通道α1C和α3亚基抑制高磷诱导的大鼠血管平滑肌细胞钙化  

Magnesium inhibits β- glycerophosphate-induced calcification of vascular smooth muscle cells by L- type calcium channel α1C and β3 in rats

作　　者：白亚玲[1] 徐金升[1] 袁景霞[1] 张俊霞[1] 崔立文[1] 张胜雷[1] 

机构地区：[1]河北医科大学第四医院肾内科,石家庄050011

出　　处：《中华肾脏病杂志》2016年第10期759-765,共7页Chinese Journal of Nephrology

基　　金：河北省自然科学基金（H2012206157）;河北省科技计划（16397733D）;河北省科技研究计划（20150351、20150310）

摘　　要：目的探讨L型钙通道（L—type calcium channel，LTCC）α1C、β3亚基在镁抑制高磷诱导的大鼠血管平滑肌细胞（VSMC）钙化中的作用。方法体外原代培养大鼠胸主动脉VSMC和血管环，用β-甘油磷酸盐（β—glycerophosphate，β—GP）诱导钙化。将细胞、血管环分别分为4组培养7d：正常对照组、高磷组（10mmol／L β—GP）、镁干扰组（10mmol／L β-GP＋3mmol／L MgSO4）、镁通道抑制剂（2-APB）组（10mmol／L β—GP＋3mmol／L MgSO4＋0．1mmol／L 2-APB）。采用茜素红染色、硝酸银染色分别检测各组VSMC、血管环的钙盐沉积情况，且用邻甲酚酞络合酮比色法检测钙含量。反转录PCR、Western印迹检测VSMC中LTCC α1C、β3亚基，Runx2的基因和蛋白表达。免疫组织化检测各组血管环中Runx2蛋白表达。ELISA测定VSMC碱性磷酸酶（ALP）活性，荧光探针法测定细胞内钙离子浓度。结果与正常对照组比较，高磷组VSMC钙含量，ALP活性，LTCCα1C、LTCCβ，和Runx2mRNA和蛋白的表达，胞内钙离子浓度均增加（均P〈0．05）；与高磷组比较，镁干扰组VSMC钙含量，ALP活性，LTCCα1C、LTCCβ3和Runx2mRNA和蛋白的表达，钙离子浓度均降低（均P〈0．05）。镁干扰组血管环组织钙含量和Runx2蛋白表达均低于高磷组（均P〈0．05）。镁通道抑制剂组以上各项目与高磷组比较差异均无统计学意义。结论镁可以抑制高磷诱导的大鼠血管钙化，其可能是通过抑制LTCCα1C和β3亚基表达，阻滞VSMC钙离子内流，降低Runx2的表达，从而抑制VSMC发生表型转化实现的。Objective To explore the effects of L-type calcium channel （LTCC） α1C and β3 subunits on that magnesium inhibited thoracic aortic calcification induced by β- glycerophosphate （β- GP）. Methods Vascular smooth muscle cells （VSMCs） and aortic rings from rat aortic were cultured, then divided into control group, high phosphorus group （10 mmol/L β-GP）, magnesium group （10 mmol/L β-GP＋3 mmol/L MgSO4） and 2-APB （an inhibitor of magnesium transporter） group （10 mmol/L β- GP＋3 mmol/L MgSO4＋0.1 mmol/L 2-APB）. Calcium deposition of VSMCs and aortic rings were respectively measured by alizarin red staining and Von Kossa staining, meanwhile the quantification of their calcium was tested by OCPC. The mRNA expressions of Runx2, LTCC α1C and β3 in VSMCs were detected by RT-PCR, and their protein expressions were detected by Western blotting. Intracellular calcium ion of VSMCs was tested by fluorescence probe and alkaline phosphatase （ALP） activity was measured by ELISA. The Runx2 expression of aortic rings was detected by immunohistochemistry. Results After VSMCs stimulated for 7 days, calcium, ALP, mRNA and protein expressions of LTCCα1C, LTCCβ3 and Runx2, and intracellular calcium ion in high phosphorus group were higher than those in control group （all P 〈 0.05）. Moreover, calcium, ALP, mRNA and protein expressions of LTCCα1C, LTCCβ3 and Runx2, and intracellular calcium ion were decreased in magnesium group as compared with those in high phosphorus group （all P 〈 0.05）. In aortic rings, magnesium group had lower calcium and protein expression of Runx2 than high phosphorus group. No statistical difference between 2-APB group and high phosphorus group was observed in above indexes （all P 〉 0.05）. Conclusion Magnesium may down-regulate expressions of LTCC α1C and β3 subunit, prevent calcium influx and then inhibit osteogenic differentiation so as to reduce β-glycerophosphateinduced VSMCs calcification.

关 键 词：镁 肌细胞 平滑肌 钙质沉着症 钙通道 L型 

分 类 号：R692[医药卫生—泌尿科学]