Klotho蛋白对血管平滑肌细胞钙化的影响  

Role of α-Klotho in calcification of rat aortic vascular smooth muscle cells

作　　者：陈天雷[1] 毛慧娟[1] 陈铖[1] 吴琳[1] 王宁宁[1] 赵秀芬[1] 钱军[1] 邢昌赢[1] 

机构地区：[1]南京医科大学第一附属医院肾内科,210029

出　　处：《中华肾脏病杂志》2015年第6期434-439,共6页Chinese Journal of Nephrology

基　　金：基金项目：“十二五”国家科技支撑计划项目（2011BA110808）;江苏省卫生厅医学科研基金（Z201002）

摘　　要：目的 探讨Klotho蛋白对高磷诱导的大鼠血管平滑肌细胞成骨样转化、钙化作用的影响及其可能的机制.方法 体外培养大鼠血管平滑肌细胞,被分为5组：对照组;钙化组：10 mmol/L β甘油磷酸诱导平滑肌细胞钙化;钙化＋LiCl组：在钙化组基础上加入5 mmol/L的LiCl;钙化＋Klotho组：在钙化培养基中加入不同浓度的重组小鼠Klotho蛋白（rm Klotho）;钙化＋Klotho＋LiCl组：在钙化＋Klotho组基础上加入Wnt/β-catenin通路激动剂氯化锂（LiCl）干预.12 d后,茜素红染色法观察细胞的钙盐沉积,分光光度计法测定细胞外基质钙含量.免疫荧光法检测α平滑肌肌动蛋白（α-SMA）的表达.Western印迹法检测骨形态形成蛋白2（BMP2）、Runt相关转录因子2（Runx2）、β连环蛋白（β-catenin）的表达.结果 β甘油磷酸诱导大鼠血管平滑肌细胞12 d后,茜素红染色结果显示钙盐沉积明显;细胞外基质钙含量增加.与对照组相比,钙化组细胞BMP2,Runx2,β-catenin表达上调,α-SMA表达下调（均P ＜ 0.05）.与钙化组相比,钙化＋Klotho组细胞钙化程度减轻,BMP2,Runx2,β-catenin表达下调,α-SMA表达上调（均P＜ 0.01）.与钙化＋Klotho组相比,钙化＋Klotho＋LiCl组细胞BMP2、Runx2及β-catenin表达上调（均P ＜ 0.01）.结论 Klotho蛋白可减轻高磷诱导的血管平滑肌细胞的成骨细胞转化、钙化作用,其机制可能与Wnt/β-catenin通路的抑制有关.Objective To investigate the role and possible mechanism of α-Klotho in calcification and osteogenic transition of cultured rat aortic vascular smooth muscle cells（VSMCs）.Method VSMC were cultured in vitro and divided into 5 groups：（1）control group; （2）β-glycerophosphate group; （3）β-glycerophosphate＋LiCl group; （4）β-glycerophosphate＋Klotho group; （5）β-glycerophosphate ＋ Klotho ＋ LiC[group.Calcium deposits were checked by Alizarin red staining.Extracellular calcium content were determined by arsenazo-Ⅲ method.The expressions of bone morphogenetic protein-2 （BMP2）,runt-related transcription factor 2 （Runx2） and β-catenin were measured by Western blotting at day 12.α-smooth muscle actin （α-SMA） was checked by fluorometry method.Results β-glycerophosphate induced a time-dose-dependent increasing of BMP2,Runx2,β -catenin expressions and calcium concentration.Alizarin red staining were positive under the condition of high phosphorus.The expression of BMP2,Runx2,β-catenin and calcium concentration were decreased after treating with rmKlotho.The expression of BMP2,Runx2 and β-catenin were upregulated when treated with LiCl.Conclusions Klotho can ameliorate the calcification and osteogenic transition of VSMCs induced by β-glycerophosphate.The mechanism of Klotho in preventing VSMCs from calcification may be partially through the inhibition of Wnt/β-catenin cellular signal pathway.

关 键 词：平滑肌细胞 血管 钙质沉着症 KLOTHO蛋白 WNT通路 骨形态形成蛋白2 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]