白细胞介素6对体外培养人脐动脉平滑肌细胞成骨样转化、钙化的影响  

Role of interleukin 6 in osteogenic transition and calcification of human umbilical artery smooth muscle cells in vitro and the possible cell signal transduction way

作　　者：孙明姝[2] 郭永平[1] 顾乐怡[1] 戴慧莉[1] 严玉澄[1] 倪兆慧[1] 钱家麒[1] 

机构地区：[1]上海交通大学医学院附属仁济医院肾内科,200001 [2]青岛大学医学院附属医院风湿免疫科

出　　处：《中华肾脏病杂志》2009年第7期548-554,共7页Chinese Journal of Nephrology

基　　金：国家自然科学基金（30370653）;上海市教委基金（04BC35）

摘　　要：目的研究白细胞介素6（IL-6）对体外培养人脐动脉平滑肌细胞（HUASMC）成骨样转化、钙化的作用及可能的信号通路。方法组织植块法原代培养HUASMC。培养基加入不同浓度重组人IL-6（rhIL-6）孵育细胞，设空白对照组。茜素红S钙沉积染色及甲氧-酚酞络合酮法检测细胞层钙盐含量。实时定量PCR、荧光定量法以及Western印迹法分别检测骨特异性碱性磷酸酶（BAP）、骨桥蛋白（OPN）、骨形成蛋白2（BMP2）和骨保护素（OPG）基因以及蛋白表达。凝胶迁移滞后实验（EMSA）检测核心结合因子α1亚基（Cbfα1）的结合活力，以及分别应用p38丝裂原活化蛋白激酶（p38MAPK）抑制剂SB203580和蛋白激酶C二氢神经鞘氨醇（DHC）后Cbfα1的结合活力。结果rhIL-6 50μg／L诱导12d，细胞基质层茜素红S染色阳性。与对照组相比，细胞层钙盐含量在rhIL-610μg／L组刺激9d[（0．76_＋0．02）mmol／g蛋白1和12d[（1．54±0．11）mmol／g蛋白]升高，50μg／L组刺激6d[（1．81±0．03）mmol／g蛋白]、9d[（2．08±0．10）mmol／g蛋白]和12d[（3．22±0．18）mmol／g蛋白]升高，并呈时间、剂量依赖地增加。rhIL-6 10μg／L刺激12h，BMP2mRNA（3．04±0．07）和蛋白（8．14±0．41）及BAPmRNA（2．51±0．11）和蛋白（3．96±0．54）表达上调；刺激72h，OPN mRNA（3．14±0．32）和蛋白（2．57±0．43）水平及OPGmRNA（4．06±0．24）和蛋白（3．46±0．34）水平上调。rhIL-6刺激6h，Cbfα1结合活力增加；DHC能够部分抑制rhIL-6诱导的Cbfctl结合活力增加，SB203580没有明显作用。结论IL-6体外能够诱导HUASMC发生钙化和成骨样转分化，这可能是临床观察到IL-6与血管钙化相关的机制之一。IL-6的这一作用可能与细胞内蛋白激酶C通路的活化有关。Objective To investigate the role of recombinant human interleukin 6 （rhIL-6） in calcification and osteogenic transition of cultured human umbilical artery smooth muscle cells （HUASMC）, and the possible cell signal transduction way. Methods HUASMCs were isolated by the explant method. HUASMCs were treated with （treatment groups） or without （control group）rhIL-6. Alizarin Red S stain was applied for calcium deposition in extracellular matrix of control cells and the cells treated with rhIL-6 50 μg/L at day 12. Calcium concentration in cell layer of control group and treatment group （treated with rhlL-6 10μg/L and 50 μg/L, respectively） was determined calorimetrically by the o-eresolphthalein complexone method at day 3, 6, 9 and 12, and corrected by total cell proteins. The mRNA expressions of bone-specific alkaline phosphatase （BAP）, osteopontin （OPN）, bone morphogenetic protein-2 （BMP2） and osteoprotegerin （OPG） were estimated by real-time PCR in 12, 24 and 72 hours. OPN, BMP2 and OPG expressions were assessed by Western blotting and the BAP concentration at the same time was checked by fluorometry method . Electrophoretie mobility shift assays （EMSA） was used to detect the binding activity of transcription factor Cbfα1 with or without inhibitors of p38-MAPK （SB203580） and PKC （DHC） after 6 hours stimulation by rhlL-6 10 μg/L. Results rhIL-6 induced a positive Alizarin Red S stain and a time-dose-dependent increasing of cell layer calcium deposition. Compared with control group, rhlL-6 10 μg/L enhanced gene expression and protein levels of BAP and BMP2 at the early time （12 and 24 hours）, and of OPN and OPG at later hours （24 and 72 hours）. RhIL-6 still induced an increasing of binding activity of Cbfctl, which could be partially blocked by DHC but not SB203580. Conclusions rhIL-6 induces HUASMCs calcification and osteogenic transition in vitro, which may be one of the mechanism involved in IL-6 associated vascular calcification as observed in clinical

关 键 词：白细胞介素6 脐动脉 肌细胞 平滑肌 核心结合因子α1亚基 血管钙化 骨形态发生蛋白质类 

分 类 号：R543[医药卫生—心血管疾病]