骨形态形成蛋白4通过受体Ⅱ／Smad信号通路对瞬时受体电位离子通道蛋白表达的影响  

Bone morphogenetic protein 4 increases canonical transient receptor potential protein expression by activating bone morphogenetic protein receptor Ⅱ/Smad signaling pathways in pulmonary arterial smooth muscle cells

作　　者：李晓岩[1] 冉丕鑫[2] 王健[2] 

机构地区：[1]广州市第一人民医院呼吸内科,510180 [2]呼吸疾病国家重点实验室广州呼吸疾病研究所 广州医科大学附属第一医院

出　　处：《中华结核和呼吸杂志》2016年第7期539-545,共7页Chinese Journal of Tuberculosis and Respiratory Diseases

基　　金：国家自然科学基金青年科学基金项目（81200037）

摘　　要：目的：观察骨形态形成蛋白（BMP）4对大鼠远端肺动脉平滑肌细胞（PASMCs）内Smad信号通路的影响，探讨其对瞬时受体电位离子通道（TRPC ）蛋白1和6的作用。方法雄性Wistar大鼠6只，体重280-300 g，显微分离远端肺动脉酶消化法原代培养高纯度PASMCs，检测BMP4对于Smad信号通路的激活，利用BMP受体Ⅱ（BMPRⅡ）的小干扰RNA（SiRNA），选择性抑制Smad信号通路，探讨BMP4／BMPRⅡ信号转导通路对TRPC1、6蛋白表达的影响，利用Western blot 技术检测蛋白表达量。结果 BMP4作用于大鼠远端PASMCs，可以迅速激活Smad信号通路，而后随时间的延长逐渐减弱。 BMPRⅡsiRNA转染大鼠远端 PASMCs，能够有效地沉默 BMPRⅡ蛋白表达， BMP4刺激转染后的PASMCs，Smad磷酸化水平较正常PASMCs低。 BMPRⅡsiRNA转染降低了BMP4对TRPC1、6蛋白的促表达作用，与转染NT－目标SiRNA相比，在转染BMPRⅡ SiRNA的PASMCs， BMP4处理后TRPC1蛋白表达与β－肌动蛋白的灰度值之比降低2.7倍（ P＜0.05）；TRPC6蛋白表达与β－肌动蛋白的灰度值之比下降2.5倍（ P＜0.05）。结论 BMP4可通过结合大鼠远端PASMCs的BMPRⅡ激活Smad信号转导途径，上调TRPC1、6蛋白的表达。Objective To observe the effect of bone morphogenetic protein 4 （ BMP4） on the Smad signaling pathway in rat distal pulmonary artery smooth muscle cells （ PASMCs ） , and to explore the role of the transient receptor potential ion channel （TRPC） protein 1 and 6（TRPC1,6） in rat distal PASMCs. Methods Distal pulmonary arteries were isolated from adult male Wistar rats （n=6, 280-300 g）.The endothelium-denuded pulmonary artery tissue was digested using Collagenase and PASMCs were cultured . Activation of BMP4 signaling pathway in Smad was detected by Western blotting .Western blotting was used for the measurement of protein to determine the involvement of BMP 4/BMPRⅡ signaling in BMP4-inducd TRPC1 and TRPC6 protein expression , and Smad signaling was inhibited by the specific BMPRⅡ small interfering RNA （BMPRⅡSiRNA）.Results Rapid phosphorylation of Smad1/5/8 was seen after 15 min of stimulation with BMP4, which was reduced with time.The BMPR Ⅱ proteins was effectively down-regulated in the PASMCs after transfection with BMPRⅡ SiRNA, and the phosphorylation of Smad 1/5/8 BMP4-induced was decreased in the PASMCs transfected with BMPR Ⅱ SiRNA compared to the normal PASMCs.In addition, transfection of BMPRⅡsiRNA also attenuated BMP4-induced TRPC1 and TRPC6 protein expression compared with transfection of NT-target siRNA in distal PASMCs.PASMCs transfected＆amp;nbsp;with BMPRⅡsiRNA showed a markedly decreased ability for BMP 4 induction as assessed by gray value ratio of Western blotting, 2.7 times and 2.5 times by TRPC1/β-actin and TRPC6/β-actin respectively, compared with NT siRNA-treated cells （ P〈0.05 ） .Conclusion TRPC1 and TRPC6 protein expression can be up-regulated through Smad1/5/8 signaling activation by combination of BMP 4 and BMPRⅡ in PASMCs.

关 键 词：骨形态形成蛋白4 SMAD蛋白质类 离子通道 BONE morphogenetic PROTEIN 4 

分 类 号：R68[医药卫生—骨科学]