生理性张应变通过增加核骨架蛋白Emerin表达抑制血管平滑肌细胞凋亡  

Physiological Cyclic Stretch Inhibits Vascular Smooth Muscle Cell Apoptosis via Inducing Expression of Nuclear Envelope Protein Emerin

作　　者：陈小虎 包晗 黄凯[1] 鲍敏[1] 张萍[1] 齐颖新[1] CHEN Xiaohu;BAO Han;HUANG Kai;BAO Min;ZHANG Ping;QI Yingxin(Institute of Mechanobiology and Medical Engineering, Shanghai Jiao Tong University, Shanghai 200240, Chin)

机构地区：[1]上海交通大学力学生物学研究所

出　　处：《医用生物力学》2018年第3期240-247,共8页Journal of Medical Biomechanics

基　　金：国家自然科学基金项目(11572199,10972178)

摘　　要：目的探讨细胞核骨架蛋白Emerin及其调控的转录因子在感受张应变力学刺激影响血管平滑肌细胞（vascular smooth muscle cells,VSMCs）凋亡中的作用。方法应用FX-5000T张应变加载系统,对体外培养的VSMCs施加5%幅度、1.25 Hz频率生理性张应变,以静止组为对照;应用cleaved-caspase3 ELISA试剂盒检测VSMCs凋亡水平,Western blotting检测VSMCs细胞核骨架蛋白Emerin蛋白表达水平。静态条件下,RNA干扰抑制VSMCs的Emerin表达,Protein/DNA芯片检测345种转录因子活性;将特异性干扰Emerin后活性发生明显变化（上调或下调超过2倍）的转录因子进行IPA（Ingenurity Pathway Analysis）信息学分析,筛选与凋亡功能相关的转录因子;染色质免疫共沉淀（chromatin immunoprecipitation,CHIP）结合q PCR检测特异性干扰Emerin对其与两种转录因子motif区域结合能力的影响。结果与静止组相比,5%生理性张应变加载24 h后,VSMCs凋亡水平显著降低,提示生理性张应变对细胞具有保护作用;5%张应变作用6、12和24 h均显著增加VSMCs的Emerin表达水平。静态条件下RNA干扰抑制Emerin表达,VSMCs凋亡水平显著增加,且10种参与细胞凋亡功能调控的转录因子活性显著上调（2倍以上）,包括CREB-BP1、p300、p55、MAX、NRF-1、STAT1、STAT3、TEF1、TR和BZP;CHIP-q PCR结果显示,Emerin特异性干扰可以显著降低Emerin与STAT家族的两个成员STAT1和STAT3的motif区域结合能力。结论生理性张应变可能通过增加核骨架蛋白Emerin表达而调控Emerin与STAT1、STAT3等多种凋亡相关转录因子motif区域结合,进而调控转录因子活性影响VSMCs凋亡。探讨张应变力学刺激调控VSMCs功能的力学生物学分子机制,对揭示血管生理稳态维持和血管病理重建的分子机制具有一定意义。Objective To investigate the mechanical response of Emerin,a nuclear envelope protein,and its role in apoptosis of vascular smooth muscle cells（ VSMCs） during cyclic stretch,and the potential effect of transcriptional factors in this process. Methods Physiological cyclic stretch with the magnitude of 5% and frequency of1. 25 Hz was subjected to VSMCs in vitro by using FX-5000 T cyclic stretch loading system. VSMCs cultured under the same conditions but without applying mechanical stretch were used as the static control. The apoptosis of VSMCs was detected by using Cleaved-caspase3 ELISA kit,and the expression of Emerin was revealed by using Western blotting. The effects of Emerin on activities of 345 kinds of transcriptional factors in VSMCs were demonstrated with Protein/DNA array after Emerin specific RNA interference（ RNAi） under static condition,and the potential transcriptional factors involved in VSMC apoptosis were analyzed with Ingenurity Pathway Analysis（ IPA）software. Furthermore,the binding abilities of Emerin to the motif of 2 kinds of apoptosis-related transcriptional factors were detected with chromatin immunoprecipitation（ CHIP） and q PCR. Results Compared with the static control,the apoptosis of VSMCs was significantly decreased by 5% cyclic stretch,which suggested a protective effect of physiological cyclic stretch. The expressions of Emerin in VSMCs was remarkably increased with 5% cyclic stretch applied for 6 h,12 h and 24 h. Specific RNAi under static condition decreased the expressions of Emerin but increased the apoptosis of VSMCs. Emerin siRNA transfection remarkably increased（ more than 2 times） the activities of 10 transcriptional factors that participated in cellular apoptosis,i. e. CREB-BP1,p300,p55,MAX,NRF-1,STAT1,STAT3,TEF1,TR and BZP. CHIP-q PCR result revealed that the binding ability of Emerin to specific mofit of STAT1 or STAT3 was significantly repressed with Emerin RNAi. Conclusions Physiological cyclic stretch could increase the expression of Emerin which mi

关 键 词：周期性张应变 血管平滑肌细胞 细胞凋亡 细胞核骨架 

分 类 号：R318.01[医药卫生—生物医学工程]