KCa3.1在碱性环境促进高磷诱导大鼠胸主动脉平滑肌细胞钙化中的作用  

Role of Ca2＋-activated K＋channels in alkalinization and β-glycerophosphate induced vascular smooth muscle cells calcification

作　　者：张胜雷[1] 徐金升[1] 杨硕[1] 白亚玲[1] 张俊霞[1] 崔立文[1] 俞殷遥[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7期519-527,共9页Chinese Journal of Nephrology

基　　金：河北省自然科学基金（H2012206157）

摘　　要：目的：探究中电导钙激活性钾离子通道（intermediate-conductance Ca^2＋-activated K＋channels ，KCa3.1）在碱性环境促进高磷诱导大鼠胸主动脉平滑肌细胞（vascular smooth muscle cells ，VSMCs）钙化中的作用及机制。方法体外培养原代大鼠主动脉平滑肌细胞和大鼠胸主动脉血管环，利用10 mmol/Lβ-甘油磷酸钠制备血管平滑肌细胞钙化、血管环钙化模型。同时使用HCl和NaHCO3调节培养基pH值，细胞、血管环被随机分为6组：正常pH7.4组、正常pH8.0组、高磷组（在高磷培养基的基础上调整pH值为7.4、7.7、8.0三个亚组）、TRAM-34（KCa3.1抑制剂）干预组（在高磷pH8.0培养基的基础上添加20 nmol/L TRAM-34）。茜素红染色法和钙含量测定法判断细胞钙化程度；von Kossa染色法和钙含量测定法判断血管环钙化程度。RT-PCR、Western印迹法检测各组细胞中调节成骨和软骨分化的关键转录因子Runx2的表达，酶联免疫吸附法测定碱性磷酸酶（ALP）活性，荧光探针法测定细胞内钙离子浓度。免疫组织化学法检测各组血管环中KCa3.1、Runx2的表达。结果体外培养VSMCs 12 d后，与正常pH7.4组相比，高磷组钙盐沉积明显增高（P＜0.05），且随着pH升高逐渐增加（P＜0.05）；与高磷pH8.0组相比，TRAM-34干预组钙盐沉积明显降低（P＜0.05）。体外培养VSMCs 4 d后，碱性环境显著促进钙离子内流（P＜0.05），增加KCa3.1、Runx2表达（P＜0.05），增加ALP活性（P＜0.05），而TRAM-34干预可显著抑制钙离子内流（P＜0.05），减少Runx2表达（P＜0.05），降低ALP活性（P＜0.05）。体外血管环培养12 d后，与正常pH7.4组相比，高磷组钙盐沉积明显增高（P＜0.05），且随着pH升高逐渐增加（P＜0.05）；与高磷pH8.0组相比，TRAM-34干预组钙盐沉积明显降低（P＜0.05）。体外血管环培养4 d后，免疫组化结果显示，碱性环境促进KCa3.1和Runx2表达（P＜0.05Objective To observe the role of intermediate conductance calcium-activated potassium channels （KCa3.1） in alkalinization and β-glycerophosphate induced vascular calcification. Methods Vascular smooth muscle cells （VSMCs） and aortic rings were obtained from rat thoracic aorta, and then randomly divided into control group （pH was provided into 7.4, 8.0）, high phosphorus groups （pH was provided into 7.4, 7.7 and 8.0, VSMCs in three groups were treated with 10 mmol/L β-glycerophosphate; HCl and NaHCO3 were used to adjust the pH） and TRAM-34 group （20 nmol/L was added into pH8.0 high phosphorus dulbecco＆#39;s modified eagle＆#39;s medium）. Calcium deposition and alkaline phosphatase （ALP） activity were measured by Alizarin red staining, calcium content and enzyme linked immunosorbent assay after cells were simulated for 12 days. Intracellular free Ca^2 ＋ was measured by ELISA. The expression of KCa3.1, runt-related transcription factor 2 （Runx2） were detected by RT-PCR and Western blotting 4 days after cells were stimulated. Calcium deposition was measured by von Kossa staining and calcium content after aortic rings were cultured for 12 days. The expressions of KCa3.1 and Runx2 were detected by immunohistochemistry after aortic rings were cultured for 4 days. Results Compared with control group, calcification in VSMCs and aortic rings were significantly increased in high phosphorus group （P〈0.05） while decreased in TRAM-34 group （P〈0.05）. Compared with control group, the expressions of KCa3.1, Runx2 and the activity of ALP in high phosphorus groups were increased （P〈0.05） while decreased in TRAM-34 group （P〈0.05）. Besides, expressions of Runx2 and KCa3.1 were augmented as the pH was higher （P〈0.05）. The expression of Runx2 in aortic rings was the same situation. Besides, the Ca^2＋ influx was blocked by TRAM-34 （P〈0.05）. Conclusions Alkalinization contributes to β-glycerophosphate induced VSMCs calcification through increase of Ca^2 ＋ influx,

关 键 词：肌细胞 平滑肌 主动脉 胸 钙质沉着症 表型转化 中电导钙激活性钾离子通道 

分 类 号：R692[医药卫生—泌尿科学]