受体相互作用蛋白激酶1在血管平滑肌细胞表型转换中的作用研究  

Research on Potential Role of Receptor-interacting Protein Kinase 1 in Phenotype Switching of Vascular Smooth Muscle Cells

作　　者：包勤学 陈丽[1] 吴思媛[2] 赵明月[2] 吴文超[2] 付华[1] 刘小菁[1,2] 

机构地区：[1]四川大学华西医院心内科,成都610041 [2]四川大学华西医院再生医学研究中心心血管疾病研究室,成都610041

出　　处：《生物医学工程学杂志》2016年第4期719-728,共10页Journal of Biomedical Engineering

基　　金：国家自然科学基金资助项目(11072163;11372204);高等学校博士学科点基金资助项目(20120181110012);四川省科技厅资助项目(2010FZ0092)

摘　　要：血管平滑肌细胞(VSMCs)表型转换在多种血管疾病中起着重要作用,因此本研究旨在探索受体相互作用蛋白激酶1(RIPK1)在血管平滑肌细胞表型转换中的表达变化及其可能起到的作用。通过血管紧张素Ⅱ(AngⅡ)刺激建立VSMCs表型转换模型,分别采用定量PCR、Western blot检测VSMCs表型转换及分泌标志物、RIPK1的表达和核因子-κB(NF-κB)的P65亚基磷酸化水平的变化,5-乙炔基-2'脱氧尿嘧啶核苷(EdU)掺入法检测细胞增殖变化,并用划痕实验检测细胞迁移情况。同时,应用RIPK1特异性小分子抑制剂Necrostatin-1(Nec-1),以及特异性RIPK1-siRNA抑制RIPK1的表达,观察RIPK1在VSMCs表型转换中的作用。结果显示,AngⅡ诱导VSMCs发生表型转换后,RIPK1表达及P65磷酸化的水平明显增加。给予Nec-1预处理或是利用RIPK1-siRNA沉默RIPK1基因后,P65的磷酸化水平呈现下调,同时能部分逆转VSMCs的表型转换并抑制其分泌、增殖和迁移能力。实验表明,AngⅡ能诱导RIPK1表达上调,同时RIPK1可能通过促进NF-κB的P65亚基磷酸化参与了VSMCs的表型转换过程。Vascular smooth muscle cells（VSMCs）phenotype switching plays an essential role in the pathogenesis of various vascular diseases.The present study aims to investigate the role of receptor-interacting protein kinases 1（RIPK1）in VSMCs phenotypic switching induced by AngiotensinⅡ（AngⅡ）.Expression of mRNA and protein of RIPK1,markers of VSMCs phenotypic switching and secretion,phosphorylation of the P65 subunit of NF-κB were measured by real-time PCR and Western blot.Meanwhile,EdU incorporation assay and wound scratch assay were performed to determine the cell proliferation and migration respectively.At the same time,Necrostatin-1（Nec-1,an known RIPK1inhibitor）and RIPK1-specific small interference RNA（siRNA）were used to inhibit the expression of RIPK1.The experimental data demonstrated that the mRNA and protein levels of RIPK1 and P65phosphorylation were increased significantly in the process of VSMC phenotypic switching induced by Ang II.Moreover,the expression of RIPK1 and P65phosphorylation were significantly down-regulated in VSMCs pretreated with Nec-1or transfected with RIPK1-siRNA.Furthermore,the proliferation,secretion and migration of VSMCs were also markedly suppressed after inhibition of RIPK1 by Nec-1or its specific siRNA.The results suggested that RIPK1 might be involved in VSMC phenotypic switching induced by Ang II,which was possibly via up-regulating the NF-κB signaling pathway.

关 键 词：受体相互作用蛋白激酶1 血管平滑肌细胞表型转换 血管紧张素Ⅱ 核因子-ΚB 

分 类 号：R54[医药卫生—心血管疾病]