磷酸钙晶体通过BMP-2／Smad信号通路促进血管钙化  

Calcium phosphate crystals promotes vascular calcification through BMP- 2/Smad signaling

作　　者：刘曜蓉[1] 严豪[1] 李振元[1] 宫丽 杨晓晓[1] 曹励欧[1] 倪兆慧[1] 钱家麒[1] 方炜[1] Liu Yaorong;Yan Hao;Li Zhenyuan;Gong Li;Yang Xiaoxiao;Cao Liou;Ni Zhaohui;Qian Jiaqi;Fang Wei(Department of Nephrology,Renji Hospital,School of Medicine,Shanghai Jiao Tong University,Shanghai Center for Peritoneal Dialysis Research,Molecular Cell Lab for Kidney Disease,Shanghai 200127,China)

机构地区：[1]上海交通大学医学院附属仁济医院肾脏科 上海市腹膜透析研究中心分子细胞(肾病)实验室,200127

出　　处：《中华肾脏病杂志》2018年第8期608-615,共8页Chinese Journal of Nephrology

基　　金：国家自然科学基金（81670691、81370864）;上海市科委资助项目（114119a5900）;上海交通大学医学院“研究型医师”队伍

摘　　要：目的探讨高磷诱导的磷酸钙晶体是否通过BMP-2／Smad信号通路激活人主动脉平滑肌细胞（HASMCs）成骨转分化。方法高磷培养基在37℃孵育3d，超速离心法分离磷酸钙晶体和上清液，扫描电镜和能量色散X射线光谱仪分析晶体理化特征。体外培养HASMCs，分为高磷组、对照组、晶体组和上清液组。茜素红染色和邻甲酚酞络合酮法测钙化结节和细胞内钙含量。Western印迹法测骨形态发生蛋白2（BMP-2）、转录因子RUNX2、骨桥蛋白（OPN）、磷酸Smadl／5／9（p-Smadl／5／9）蛋白表达。HASMCs分别进行BMP-2和Smadl基因敲减，Western印迹法测蛋白表达。结果高磷培养基诱导磷酸钙晶体形成。与对照组相比，磷酸钙晶体促进HASMCs钙化结节形成，增加细胞内钙含量，上调BMP-2、RUNX2和OPN蛋白表达（均P〈0．05）。磷酸钙晶体刺激HASMCs 30min后，p-Smadl／5／9蛋白水平达到峰值（P〈0．05）。敲减BMP-2基因后，磷酸钙晶体刺激HASMCs后p-Smadl蛋白表达受抑，RUNX2和OPN蛋白表达降低（均P〈0．05）。敲减Smadl基因后，磷酸钙晶体刺激HASMCs后RUNX2和OPN蛋白表达降低（均P〈0．05）。结论高磷诱导的磷酸钙晶体通过BMP-2／Smad信号通路促进HASMCs成骨转分化。Objective To investigate the role of BMP- 2/Smad signaling pathway in the osteogenic differentiation of human aortic smooth muscle cells （HASMCs） caused by hyperphosphatemia -induced calcium phosphate （CAP） crystals. Methods High- phosphate medium was incubated at 37~C for 3 days. CaP crystals and supernatant were isolated by ultracentrifugation. Scanning electron microscope and energy dispersive X- ray spectroscopy were performed for analysis of physicochemical characteristics of CaP crystals. HASMCs were cultured in vitro, and divided into high- phosphate, control, crystals and supernatant groups. Calcification was visualized by Alizarin red staining. Calcium loads in cells were quantified by ocresolphthalein complexone method. Protein expression of bone morphogenetic protein- 2 （BMP-2）, Runt-related transcription factor 2 （RUNX2）, osteopontin （OPN）, phospho-Smadl/5/9 （p-Smadl/5/9） were quantified by Western blotting. After knockdowns of BMP-2 and Smadl with small hairpin RNA （shRNA） interfering respectively in HASMCs, protein expressions were measured by Western blotting. Results High-phosphate medium induced the formation of CaP crystals. Compared with the cells in control group, CaP crystals significantly induced HASMCs calcification, increased calcium loads and up-regulated the levels of BMP-2, RUNX2 and OPN proteins （all P 〈 0.05）. After the addition of CaP crystals into HASMCs, the level of p- Smad 1/5/9 protein peaked at 30 min （P 〈 0.05）. After BMP-2 was knocked down in HASMCs, the expression of p-Smadl caused by CaP crystals was blocked completely, and the expressions of RUNX2 and OPN caused by CaP crystals were reduced significantly （all P 〈 0.05）. After Smadl was knocked down in HASMCs, the expressions of RUNX2 and OPN caused by CaP crystals were decreased significantly （all P 〈 0.05）. Conclusions Hyperphosphatemia-induced CaP crystals promoted osteogenic differentiation of HASMCs through the BMP-2/Smad signaling pathway.

关 键 词：肾功能不全 慢性 钙质沉着症 磷酸钙类 肌细胞 平滑肌 

分 类 号：R692[医药卫生—泌尿科学]